J Chromatogr A - 2009 1216 2 29

Journal of Chromatography A, 1216 (2009) 229
Contents lists available at ScienceDirect
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Review
Sample preparation for the analysis of isoavones from soybeans and soy foods
M.A. Rostagno a, , A. Villares a , E. Guillamn a , A. Garca-Lafuente a , J.A. Martnez a,b
a
Centro para la Calidad de los Alimentos, Instituto Nacional de Investigacin y Tecnologa Agraria y Alimentaria (INIA),
Campus Universitario Duques de Soria, 42004 Soria, Spain
b
Universidad de Navarra, Dpto. Fisiologa y Nutricin, Edicio de Investigacin, C/Irunlarrea, 1, 31008 Pamplona, Spain
a r t i c l e
i n f o
Article history:
Received 6 August 2008
Received in revised form 3 November 2008
Accepted 13 November 2008
Available online 19 November 2008
Keywords:
Reviews
Isoavones
Soybeans
Sample conservation
Sample preparation
Extraction
Analysis
a b s t r a c t
This manuscript provides a review of the actual state and the most recent advances as well as current
trends and future prospects in sample preparation and analysis for the quantication of isoavones
from soybeans and soy foods. Individual steps of the procedures used in sample preparation, including sample conservation, extraction techniques and methods, and post-extraction treatment procedures
are discussed. The most commonly used methods for extraction of isoavones with both conventional
and modern techniques are examined in detail. These modern techniques include ultrasound-assisted
extraction, pressurized liquid extraction, supercritical uid extraction and microwave-assisted extraction.
Other aspects such as stability during extraction and analysis by high performance liquid chromatography
are also covered.
2008 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
6.
7.
8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General aspects of soy isoavones determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction techniques and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Solid and semi-solid samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.1.
Conventional extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.2.
Modern extraction techniques and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Liquid samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Optimization of extraction conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Critical comparison of extraction methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-treatment of extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Separation approaches/techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
4
4
6
7
7
7
12
21
22
23
25
26
27
28
28
Abbreviations: , Dielectric constant; ACE, Acetone; ADi, Acetyl daidzin; AGi, Acetyl genistin; AGly, Acetyl glycitin; ASE, Accelerated solvent extraction; CE, Capillary
electromigration techniques; De, Daidzein; Di, Daidzin; DMSO, Dimethylsulfoxide; DSM, Defatted soybean meal; EtOH, Ethanol; Ge, Genistein; Gi, Genistin; Gle, Glycitein; Gly,
Glycitin; MAE, Microwave-assisted extraction; MeCN, Acetonitrile; MeOH, Methanol; MGi, Malonyl genistin; MDi, Malonyl Daidzin; MGly, Malonyl glycitin; PLE, Pressurized
liquid extraction; PSE, Pressurized solvent extraction; SC-CO2 , Supercritical CO2 ; SFE, Supercritical uid extraction; SPE, Solid phase extraction; SPI, Soy protein isolate; SPME,
Solid phase microextraction; SWE, Superheated water extraction; UAE, Ultrasound-assisted extraction.
Corresponding author. Tel.: +34 975 233204; fax: +34 975 233205.
E-mail address: rostagno.mauricio@inia.es (M.A. Rostagno).
0021-9673/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.11.035
M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229
1. Introduction
Functional foods are one of the most promising elds concerning
nutritional sciences. These food-stuffs are interesting from the consumer point of view with the prospect of maintaining health and
preventing diseases by using natural foods as part of the habitual
diet, and also from the industry point of view, for the added value
of the products. There are several raw materials that can be used for
healthy purposes and soybeans are among those with the greatest
potential. Soybeans are one of most produced and commercialized
commodities worldwide. Actually, there are several foods derived
or based on soybeans such as soy milk, tofu and tempeh, and the
consumption and use of soybeans (texturized soy protein, concentrated soy protein and soy protein isolate) as additives by the food
industry is increasing every year [14].
The potential of soybeans as a functional food is being currently
explored by the food industry. Indeed, soybeans and soy foods, like
soymilk, tofu, miso and tofu, are widely promoted and eaten based
on assumed relationships between its consumption and benecial health effects in humans including chemoprevention of breast
and prostate cancer, osteoporosis, cardiovascular disease as well
as relieving menopausal symptoms. Evidence provided not only by
epidemiological studies showing a lower incidence of these health
conditions in Asian countries like Japan and China, which have high
soy consumption, but also from intervention studies, is the basis of
this relationship [512].
During the last decades our knowledge about the dietary impact
on health and well-being has been highly increased and often
related to specic food components. Several classes of phytochemicals have been identied in soybeans, including protease inhibitors,
phytosterols, saponins, phenolic acids, phytic acid and isoavones
[1316]. Of these, isoavones are particularly noteworthy because
soybeans are the only signicant dietary source of these compounds. Isoavone content in soybeans can range from 0.4 mg to
9.5 mg of total isoavones per gram, which can be inuenced by
genetics, crop year and growth location [1719]. More importantly,
these compounds have shown several in vitro and in vivo benecial
properties consistent with the potential soybean effects on health.
There are several possible mechanisms of action by which
isoavones may act on disease prevention, including estrogenic/
anti-estrogenic activity, cell anti-proliferation, induction of cellcycle arrest and apoptosis, prevention of oxidation, antiinammatory, regulation of the host immune system, and changes
in cellular signaling [7,2028]. The actual mechanisms in the human
organism have not been fully established and metabolism may play
an important role. Furthermore, besides of evidence of available
epidemiological or intervention studies and in vitro observations,
there are several reports indicating that several of the specic
potential soybean health benets are linked to isoavone intake
[8,2932].
However, there is still controversy and an unanimous position
about if isoavones, other soy phytochemicals or components are
responsible for the health benets of soy consumption is still far
from being reached. Because the data in humans are not conclusive for any of these possible benets, it is important to conduct
more studies investigating isoavones and soy foods in the diet to
health outcomes. An accurate food composition database is crucial for such studies. That is the reason why there is an increasing
interest of scientists focused in developing newer extraction and
analysis methods for the characterization of soybean functional
components, especially isoavones, and about the relationships
between their consumption and benecial health effects in
humans.
Isoavones are a subclass of avonoids and are also described
as phytoestrogen compounds, since they exhibit estrogenic activ-
Fig. 1. Chemical structures of soybean isoavones and abbreviations.
ity (similar effects to estradiol hormones). The basic characteristic

isoavone structure is a avone nucleus, composed by two benzene rings (A and B) linked to a heterocyclic ring C (Fig. 1). The
benzene ring B position is the basis for the categorization of
the avanoid class (position 2) and the isoavonoid class (position 3). The main isoavones found in soybeans are genistein
(4 ,5,7-trihidroxyisoavone), daidzein (4 ,7-dihidroxyisoavone),
glycitein (4 ,7-dihidroxy-6-metoxi-isoavone) and their respective
acetyl, malonyl and aglycone forms (Fig. 1) [3339]. Biochanin A and
formononetin (which are derivatives of genistein and daidzein) are
generally less abundant in soy than the 12 main forms and which
are found mostly in clover and alfalfa sprouts [40].
Isoavone content of available soy foods in several countries
is been intensively investigated. Quantication of isoavones in
the soybeans and soy foods consumed in the USA [4044], Japan
[45,46], Italy [47], UK [48,49], Singapore [43,50], Australia [51],
Indonesia [50,51], Brazil [52], and Canada [53] have been published
in the last decade.
Besides of individual reports, there are food composition
databases and compilations from these values specically focusing on isoavone distribution [5462]. These reports supply useful
information to investigators determining the intake of phytoestrogens in order to relate intakes to potential biological activities. Also,
they can be used by health professionals and consumers to estimate
individual phytoestrogens intake and design personalized diets in
order to achieve biologically active concentrations of these functional compounds.
When the intake of isoavones is estimated, the quality of the

food composition database is important. This is critical in the case
of foods consumed regularly, in large quantities, or containing
ingredients with concentrated amounts of phytoestrogens. Future
analyses of the isoavone content of basic ingredient foods and
commercial items commonly consumed in the diet will enable
more accurate estimates of phytoestrogen intake and obtain reliable conclusions about their role on health [56,58,63].
Due to the enormous efforts done in the last few years to evaluate isoavone composition in foods and its relation with nutritional
issues and health effects it is of ultimate importance to develop reliable and precise methods for the quantication of these compounds
in foods. Because of the increasing complexity of the food supply,
there are major challenges in collecting reliable food consumption data for phytoestrogen intake estimates. Several extraction
methods have been used for quantication purposes without adequate validation of the extraction procedure and far from optimized
extraction conditions, which can lead to erroneous measurements
and calculations. Besides, optimal extraction conditions can be used
to save time, resources and provide reliable information. Moreover, only scattered data are available in the scientic literature
and a review of the subject is needed to provide essential information on the topic and to identify future research elds of action.
Therefore, the aim of the present manuscript is to provide a critical review of the actual state, the most recent advances as well
as current trends and future prospects in sample preparation and
analysis for the quantication of isoavones from soybeans and
related foods.
2. General aspects of soy isoavones determination
The four common steps for any analytical method are sampling, sample preservation, sample preparation and analysis. Fig. 2
presents a general overview of the most common steps for sample
preparation for the determination of soy isoavones.
The initial step in any analysis is sampling, where a representative sample is collected from the entire sample matrix that needs
to be analyzed. The entire food-stuff should be represented in the
sample that will be used for the analysis. Sample preservation is
an important step as there is often some delay between sample
collection and/or preparation and analysis. Proper sample preservation ensures that the sample retains its physical and chemical
characteristics from the time it is collected to the time it is analyzed.
Sample preparation may consist of multiple steps such as
drying, homogenization, sieving, extraction of target compounds,
pre-concentration, hydrolysis and derivatization. Sample preparation can seek several objectives: to increase the efciency of an
assay procedure, to eliminate or reduce potential interferences, to
enhance the sensitivity of the analytical procedure by increasing
the concentration of the analyte in the assay mixture, and sometimes to transform the analyte of interest to a more suitable form
that can be easily separated, detected, and/or quantied. Isoavone
determination is complex since its concentration in the sample
depends of several variables which may difcult the determination.
Overall, the ultimate goal is to obtain a concentrated extract with
all isoavones and free of interfering compounds from the matrix
[6466].
The quantication of isoavones in solid samples is usually performed by extracting isoavones from the food matrix using a
certain solvent and then analyzing the extract by one of the several analysis techniques available, including gas chromatography,
high performance liquid chromatography (HPLC) and immunoassay, among others. The most used analysis technique is, without
doubt, reverse-phase HPLC using C18 based columns with water
and methanol or acetonitrile containing small amounts of acid as
the mobile phase.
The extraction phase is extremely important and the process
will depend of analyte liberation from the matrix, which will allow
quantitative determinations of target compounds. Moreover, the
extract should mimetic the original isoavone composition and
prole as much as possible. For the efcient extraction several
parameters should be dened like the solvent, temperature, sample
amount and time.
Optimization of the extraction conditions is normally accomplished using the classical one-variable-at-a-time method, in which
the optimization is directly assessed by systematic alteration of
one variable, while the others are kept constant. Some authors
use experimental designs for the determination of interactions
between parameters and selecting the most suitable extraction
conditions while minimizing the number of experiments. In the
experimental design strategies the values of all the factors under
study are varied in each assay in a programmed and rational way. It
is thus possible to detect the inuencing factors while the number
of trials can be kept to a minimum [67,68].
3. Sample stability
Fig. 2. Most common steps for sample preparation for the determination of soy
isoavones.
In analytical practices, the importance of sample conservation

must be emphasized. Indeed, if not carefully controlled can lead to
errors that cannot be corrected afterwards since will consequently
affect the outcome of the nal analysis. Thus, the results obtained,
instead of being the source of information, can produce misinformation.
Often too little attention is given to the handling of soybean, soy
foods or isoavone extract samples after their collection and before
the actual instrumental analysis. How and for how long different
samples can be stored to preserve their original isoavone prole
is particularly important since some isoavones have a relatively
unstable character. Chemical changes of isoavone structures have
been reported to occur during the processing of soybeans and
soy products. The most frequently observed chemical changes of
isoavones during the processing are decarboxilation of malonyl
glucosides to acetyl glucosides and ester hydrolysis of malonyl
and acetyl glucosides to underivatized glucoside. It is also possible
Fig. 3. Most common possible degradation paths of soybean isoavones.
for all different conjugated forms to generate the aglycone forms

by cleavage of the glucosidic bond [6973]. An overview of the
most common possible degradation paths of soy isoavones are
presented in Fig. 3. However, only there are only a few studies
about isoavone stability during storage of soybeans, soy foods and
extracts. In fact, only recently the stability of isoavones in soybeans
stored under different conditions was investigated [7476].
Information from the few reports available indicates that storage
of soybeans and soy foods for prolonged times at room temperature
can affect isoavone distribution and content. Generally, the concentrations of individual isoavones either signicantly decrease
or increase during storage for long periods. With storage, malonyl
glucosides concentration tends to decrease while concentration
of glucosides and aglycones tend to increase. Concentration of
malonyl glucosides can decrease by about 2 times, whereas glucosides and aglycone concentration can increase up to 34 times
during storage for 2 years [74]. However, storage at room temperature may, in some cases, decrease glucoside and aglycone content
[75].
Moreover, not only the isoavone prole may be affected by the
course of time, but also total isoavone concentration, especially
in the rst year of storage. Afterwards, storage (up to 2 years) only
slightly changes total isoavone content but still affects isoavone
prole of the samples [74]. Storage for prolonged periods reduces
total isoavone concentration and the reduction level depends
of the soybean cultivar. While some cultivars show only a slight
decrease on total isoavone concentration, others present a severe
decrease on concentration of these compounds [75]. Furthermore,
the level and type of the modication on isoavone prole and
losses caused by storage may be dependent of temperature, relative
humidity and the soybean cultivar among other factors.
The variation on isoavone concentrations were positively correlated with storage temperature and total isoavones were related
with the amount of malonyl glucoside and glucoside groups. Storage at low temperature can result in changes in isoavone levels
similar to those observed during processing [75] or may not affect
isoavone distribution [76]. Endogenous glucosidases, humidity
and inuence of soybean variety as observed by Kim et al. [75] may,

partially, explain differences observed in these studies.
Relative humidity as well as temperature can inuence the
changes on isoavone prole during storage. Storage of soybeans under high relative humidity (84%) and temperature (30 C)
conditions for extended periods of time (9 months) causes the interconversion between aglycones and -glucosides. Storage under
these conditions can affect isoavone prole to a point that
the major constituents can become the minor constituents, and
vice-versa. Storage under milder storage conditions (57% relative
humidity and 20 C) causes only the interconversion between glucosides and malonyl glucosides [76].
It has also been demonstrated that some isoavones in soymilk
are subjected to degradation [77] during storage. For example, Gi is
labile to degradation during storage at room temperature, although
at a low rate. Losses of Gi with time showed typical rst-order kinetics and increased with storage temperature. The Di concentration
was not inuenced by storage between 15 C and 37 C. However,
degradation of Di was not discarded, since it was possible that
a combination of deacetylation of ADi to Di and Di degradation
was taking place simultaneously. At early stages of soymilk storage at 8090 C, ADi concentration increased, followed by a slow
decrease. However, malonyl isoavones, which are more sensitive
to degradation, were not studied.
Therefore, more research it is still needed on the effects of
storage environments, such as humidity and temperature, on the
transformation and losses of isoavone groups. The characterization of the differences between soybeans cultivars related with
the change of isoavones, with special emphasis on endogenous
glucosidases and to identify suitable conservation methods are
important pending tasks. Also, more research aimed at different
soy products is required in the same direction.
Finally, it is imperative that authors conducting quantication
studies be specic about sample conservation aspects. It must be
clear for how long the soybean or soy food sample have been stored
before actual analysis and the conditions such as temperature,
humidity, etc.
Also, while studying isoavone proles and distribution in

foods and over different cropping years it is highly recommendable to perform the determinations after harvest, and not analyze
all the samples at the same time (after all samples were harvested), with the inherent differences and errors caused by storage
after 1 year or more, even if samples are stored at low temperature. It is also recommendable to refer isoavone content to dry
weight since variation of sample humidity may inuence concentration.
On the other hand, storage of samples after extraction and before
analysis can also affect isoavone proles and result in avoidable
analytical errors. Due to the relatively unstable character some
isoavones as well as by the action of native -glucosidases, resulting in a rapid degradation or interconversion between chemical
forms, quantication of isoavones is a complicated procedure. The
most susceptible to degradation isoavones seems to be the malonyl forms. Barnes et al. [78] noted that isoavones in 80% MeOH
extracts of soy samples kept at room temperature were converted
gradually from malonyl glucosides to -glucosides. Coward et al.
[71] reported a slight conversion of the malonyl glucosides to the
-glucosides conjugates at room temperature and that malonyl glucoside conjugates are stable at 4 C for 24 h, but prolonged storage
also causes conversion to the -glucosides conjugates.
Later, Murphy et al. [42] reported a conversion rate of
0.20.3 mol% per hour of malonyl forms to glucosides in soy
isoavone extracts at room temperature. Evidences show that
prompt analysis of the extracts after extraction or other strategies,
such as maintenance of auto sampler at low temperatures (45 C)
are necessary to minimize degradation of malonyl isoavones.
Although these procedures can elude potential analysis errors it
is essential to consider the stability of soy isoavones extracts under
storage conditions to allow better planning of routine analysis of
large number of samples and avoid analytical errors due to degradation and conversion between forms (i.e. malonyl to glucosides,
malonyl to acetyl glucosides, etc.) [79].
In one of the few published reports dealing specically with the
storage of soy isoavone extracts, Rostagno et al. [79] evaluated the
inuence of several factors (temperature, storage time, head space
and UV light) on short-term stability of samples kept on HPLC vials.
The conclusion was that samples can be stored up to 1 week with
no signicant degradation if kept at temperatures lower than 10 C
and protected from light.
On the other hand, Rijke et al. [80] evaluated the stability of
isoavone extracts obtained from red clover and observed that samples can be stored up to 2 weeks at 20 C and if samples are kept
at room temperature or if are stored dry at 20 C, degradation
starts almost immediately. Curiously, they also observed that in LC
separated fractions, red clover malonyl isoavones are more stable
when stored at low temperature after evaporation to dryness.
Aside the fact that the report did not include most common
isoavones present in soybeans it indicates that more research is
needed to nd more suitable sample conservation methods and to
evaluate longer storage of soy isoavone extracts under different
conditions before analysis.
4. Hydrolysis
As previously discussed, there are different isoavone chemical
structures, and interconversion can occur between forms depending of storage, processing and extraction conditions. Not only
sample preparation is complicated, but also the analysis step may
be critical. The accurate quantication of the total content of
isoavones is hampered by the feasibility of chromatographically
separating all the possible forms of these compounds and to nd
the corresponding reference standards. Some isoavones are par-
ticularly difcult to separate from each other (i.e. MGi, AGly and
De) [81], while others (i.e. malonyl and acetyl isoavones), due
their relative unstable character, are not widely commercially available. Coelution of other substances present in the extracts may also
add difculty to the troublesome determination of soy isoavones.
Furthermore, some isoavones might occur in as yet unidentied
forms.
A possible solution to these analytical problems is to perform
adequate sample treatment involving hydrolysis in order to reduce
the number of isoavone chemical forms occurring in the sample.
The hydrolysis procedure itself can be carried out before, during
or after the extraction using different conditions and agents. There
are two main procedures to perform the hydrolysis of isoavones
reported in the literature, basic or acidic hydrolysis. Basic hydrolysis
act on ester bonds, removing acid groups that are linked to the sugar
moiety of the isoavone glucosides. As a result, the malonyl and
acetyl glucoside isoavone forms are converted to their respective
glucosides. Acid hydrolysis breaks the bond between the isoavone
and the glucoside moieties, transforming all the isoavone derivatives, into their aglycone forms [82].
Although reaction times and temperatures for the acidic hydrolysis conditions vary a great deal, these procedures usually involve
treating the extract or food sample itself with inorganic acid (HCl)
at high temperatures in aqueous or alcoholic solvents with reaction
times ranging from a few minutes to several hours. Basic hydrolysis
entails treating the sample with a solution of NaOH and allowing
standing at room temperature from a few minutes to overnight
[8287].
Hydrolysis through the use of enzymes or a combination of
enzyme and acid [88,89] has also been used, although these
methods are less frequently used than acid or basic hydrolysis. The enzymatic hydrolysis consists of incubating the sample
with enzymes for long periods of time, ranging from a few hours
to overnight. Different enzymes have been used for the hydrolysis of isoavones, including endougenous soy -glucosidases,
-glucuronidases, sulfatases and cellulases. Similarly to basic and
acid hydrolysis, conditions vary a great deal and several different
methods have been reported [8893].
There are advantages and disadvantages with the use of
hydrolytic methods. The most obvious disadvantage is the inclusion
of an additional step, with the inherent complication of the sample
preparation procedure and the possible added analytical variability. Also, there is indication that Ge is not entirely stable under acid
hydrolysis conditions [93]. The limited information obtained using
hydrolytic methods can also be decisive, since only a few chemical forms are quantied, while using non-hydrolytic methods full
information can be accessed.
Although the hydrolysis step creates new questions with respect
with sample preparation, analyte stability and recoverability, it
greatly simplies the analysis by reducing the number of derivatives. The chromatographic analysis time is considerably shorter
and separation of target compounds is easier since there are fewer
compounds occurring in the sample. Acid hydrolysis results in the
inclusion in the quantication of isoavones that are linked to sugars other than glucose, and of glucosides of isoavones that are
not commercially available or difcult to acquire. Acid hydrolysis
is useful for the analysis of complex samples, and may be used
to identify sugar-isoavones by comparison of these results with
those from basic hydrolysis. The analysis of acid hydrolyzed extracts
is preferred when analyzing samples of unknown origin, because
it includes in the quantication the glucoside derivatives of all
isoavones available only as aglycones [82].
Moreover, the use of hydrolytic methods may reduce the analytical variability caused by stability issues during extraction since the
most unstable isoavones (malonyl glucosides) are not quantied
as such. However, it is of crucial importance when using hydrolytic

methods that authors make the necessary corrections and normalize the results by molecular weight to the aglycone forms, since the
molecular weight of the glucosides is greater than aglycones, and
therefore reported total isoavone amount can be signicantly less
than the value of non-normalized data.
Although the available evidence in the literature suggests that
the biological effects of soy isoavones depend upon aglycone form,
for analysis of soy foods for isoavonoids, the recent trend has
been to avoid hydrolysis. Using non-hydrolytic methods provide
valuable information about the exact distribution of all chemical
forms present in soybeans and soy foods. The different isoavones
may have differing pharmacokinetics and bioactivity [9496] and
this may be a key factor in understanding their biological effects.
Another logical reason to avoid hydrolysis is to minimize sample
handling, simplifying the extraction and overall analytical procedure and to shorten, as much as possible, the time required from
sampling to actual analysis. However, it is important to highlight
that the valuable information about the total isoavone concentration provided by hydrolytic methods is an essential screening
measurement and that isoavone proles are very important in an
advanced metrological step.
5. Extraction techniques and methods
5.1. Solid and semi-solid samples
Optimal solidliquid extraction involves the intimate contact
between a solid material, usually nely grinded, and a solvent that
has a maximal solubility for the analyte of interest and a minimal
solubility for the matrix, using additional external forces and heating to speed up the extraction process. Solid soy samples, such as
soybeans and soy protein, require only grinding before extraction,
but sometimes are freeze-dried to provide a homogenous powder.
Liquid samples are most often freeze-dried and also treated as solid
samples. Common methods for the extraction of the isoavones
from solid samples include organic solvent extraction with pure or
aqueous methanol (MeOH), ethanol (EtOH), acetonitrile (MeCN) or
acetone (ACE) with and without the addition of small amounts of
acids using simple soaking, mixing, shaking or soxhlet extraction.
The extraction time may range from 2 h to 24 h and the extraction
temperature from 4 C to 80 C.
More recently, modern extraction methods, such as ultrasonically assisted extraction (UAE), pressurized liquid extraction (PLE),
supercritical uid extraction (SFE) and microwave-assisted extraction (MAE) have been used for the extraction of soy isoavones
using similar solvents. In many cases, besides of ltration and centrifugation, further purication and/or pre-concentration of the
target compound fraction is applied. In these cases, evaporation
to dryness and re-dissolution on another solvent or solid phase
extraction (SPE) are the most commonly used methods. Another
common procedure during sample preparation is the hydrolysis
after the extraction (see Section 4).
5.1.1. Conventional extraction methods
Among the conventional extraction techniques soxhlet, shaking, and stirring are the most commonly used for the extraction
of isoavones from soybeans and soy foods. There are numerous
available extraction methods using these techniques with different conditions, and most of them without an appropriate method
optimization.
Several parameters can inuence the extraction of organic compounds such as polarity and amount of the solvent, temperature,
mass and kind of sample and extraction duration. In the specic case of isoavones, optimum solubility of the analyte in the
extraction solvent, one of the key parameters of any extraction

method, is very difcult to achieve since there are several chemical forms with different solubility coefcients in a given solvent.
Most methods so far developed evaluate different solvents trying
to reach an optimal condition where extraction of all isoavones is
maximized. Although abundant research on soy isoavones quantication is available only a few reports deals with the development
and optimization of extraction methods for quantication studies.
An overview of developed methods and evaluated parameters using
conventional techniques for the extraction of isoavones from soybeans and soy foods is presented in Table 1.
One of the rst studies about the extraction of soy isoavone was
published by Eldridge [97], where pure MeOH and EtOH and with
different water proportions, Ethyl acetate and MeCN were evaluated for reuxing extraction of isoavones from defatted soybeans.
From the evaluated solvent systems, 80% MeOH gave the highest isoavone extraction yields and the most reproducible results.
Using this solvent, 4 h seemed to be sufcient for extracting the
isoavones from soybean meal and no signicant differences in
the extraction efciency was reported when using different solvent:sample ratios (14:1 and 45:1). Once extraction conditions
were established, the method was used for the determination of
isoavones from soybean ours, protein concentrates and isolates.
The same method was also used for the study of the effect of environment and variety on the composition of soybean isoavones
[98].
Another pioneer study about the extraction of isoavones was
carried out by Murphy [99], who compared several solvents systems (MeOH, ACE, MeCN, and chloroform-MeOH) for the extraction
of isoavones from toasted defatted soy akes using wrist-action
shaker. The results indicated that extraction with pure solvents gave
low yields and that the addition of water or acid greatly improve the
extraction efciency of all isoavones examined (Gi, Ge, Di and De).
In terms of total isoavones and coextractives, MeCN with water or
acid was more efcient for the extraction of isoavones all other solvents systems examined and no marked difference between these
two solvents was observed in terms of total isoavones.
As a result of these two pioneer studies, 80% methanol and acidied 83% acetonitrile became the most commonly used extraction
solvents in isoavone analysis. The method developed by Murphy
[99] has been extensively used with slight modications in sample amount, solvent volume, addition of water to the extracting
solvent and shaking technique [1719,41,42,72,74,100103]. However, these slight modications of the method have an important
impact on extraction efciency and should not be used lightly, since
extraction conditions require optimization for each different sample.
As an example, Song et al. [101] reevaluated the method by Murphy [99] and reported that using water in addition to HCl and MeCN
increased recovery. For different soy samples different amounts
of water may be necessary maximize isoavone extraction. For
most soy foods, 7 mL of water was sufcient to maximize extraction using a solvent volume:sample ratio higher than 6 mL g1 .
It was also recommended that the solvent volume:sample ratio
should be adjusted for soy products with high concentration of
isoavones, particularly for isoavone supplements, which have
more than 10 mg isoavones/g. These investigators gave the example of soy germ, which have high isoavone content (>10 mg g1 ),
and reported that the normal extraction procedure would underestimate the isoavone content by 1020%. They found that adjusting
the ratio of solvent to sample weight to 95 mL g1 resulted in more
efcient extraction of isoavones from the soy germ sample.
Following the evidence of the effect of the amount of water of
the extraction solvent on isoavone extractability, Murphy et al.
[42], reevaluated the same method and conrmed that adding a
Table 1
Developed methods and evaluated parameters using conventional techniques for the extraction of isoavones from soybeans and soy foods.
Sample used for evaluation of the method
Defatted soybeans
Isoavones
Di, Gi, Gly, De, Ge and

Gle
Fixed extraction conditions

Technique: reuxing
Sample: 1 g
Solvent: 25 mL
Temperature: boiling point of
solvent
Evaluated parameters
Solvent:
EtOH, 50% EtOH, 80% EtOH
MeOH, 50% MeOH, 80% MeOH
CH3 CN
Ethyl acetate
Selected conditions
Reference
80% MeOH, 4 h
[78]
80% CH3 CN and 80% CH3 CN

(HCl)
[99]
The amount of water

optimized depending of the
sample ranged from 5 mL to
10 mL of water
[42]
Extraction time: 15 h
Technique: Wrist-action shaker
Sample: 5 g
Toasted defatted soy akes
Gi, Ge, Di and De
Solvent: 25 mL of pure solvent

or: 5 mL (H2 O or HCl
0.1N) + 20 mL (solvent)
Temperature: RT
Soy isolate, tofu, soybeans and miso
Ge, De, Gi, Di, Gly, MGi, MDi,

MGly and AGi
Technique: Stirring
Sample: 2 g,
Solvent: 1222 mL (12 mL
CH3 CN + 2 mL HCl 0.1N + water)
Solvent:
Different amounts of water
(010 mL) added to the solvent
(CH3 CN)
Temperature: RT
Toasted soy our
Soy protein
Di, Gi, Gly, De, Ge, Gle,

MDi, ADi, MGi, AGi and
MGly
Di, Gi, Gly, De, Ge, Gle, MDi,

MGi, MGly, ADi, AGi and AGly
Technique: tumbling mixer

Sample: 0.5 g
Solvent: 4 mL
Technique: rotary mixer

Sample: 1 g or amount
containing 10 mg total
isoavones (always less than
1 g)
Solvent: 17 mL
Temperature: RT
Technique: Stirring
Sample: 2 g
Soy our, tempeh, TVP and soy germ

Solvent: 19 mL (10 mL
solvent + 2 mL (HCl 0.1N or
water) + 7 mL water
Temperature: RT
Technique: Stirring
Sample: 2 g,
Solvent:
80% MeOH and 80% CH3 CN
(0.1% HCl)
Extraction time: 1, 2 and 24 h
Temperature: RT, 60 C and
80 C
Solvent:
10 mL CH3 CN + 6 mL
H2 O + 0.5 mL DMSO (IS)
10 mL CH3 CN + 2 mL HCl
0.1 M + 5 mL H2 O
80% MeOH
Water % (10100% CH3 CN)
Solvent:
53% CH3 CN, 53% ACE, 53%
EtOH, 53% MeOH
With and without acid addition
Solvent:
83% CH3 CN, 83% CH3 CN (+0.1N
HCl)
1 h,
RT
[73]
10 mL CH3 CN + 6 mL
H2 O + 0.5 mL DMSO (IS)
[104]
53% CH3 CN without

acidication
[102]
Solvent:
MeOH, 80% MeOH, 80% MeOH
(HCl)
ChroloformMeOH (90:10),
80% chroloformMeOH (90:10),
80% chroloformMeOH (90:10)
(HCl)
CH3 CN, 80% CH3 CN, 80%
CH3 CN (HCl)
ACE, 80% ACE and 80% ACE
(HCl)
Soybeans

Solvent: 12 mL
58% CH3 CN, 58% CH3 CN (+0.1N

HCl)
80% MeOH, 80% MeOH (+0.1N
HCl)
58% CH3 CN without

acidication
[105]
50% EtOH, 60 C
[106]
80% CH3 CNHCl 0.1N, 5

sequential extractions
[107]
Proposed method
[103]
99.99% EtOH,
3:1 mL g1 , 80 C and
8h
[20]
Temperature: RT
Freeze-dried soybeans
Defatted soybean meal, soy protein isolate
Di, Gi, Gly and MGi
Di, Gi, Gly, De, Ge, Gle, MDia ,

MGia and MGlya
Di, Gi, Gly, MDi, MGly, MGi, De

and Ge
Soybean our
Ge and De
Solvent:
CH3 CN (3070%)
EtOH (3070%)
MeOH (3070%)
Temperature: 10 and 60 C
Technique: Shaking
Sample: 2 g
Solvent: 10 mL
Solvent:
80% CH3 CNHCl 0.1N
80% MeOH
Temperature: RT
80% EtOH
Number of extractions: 1 and 5
Technique: homogenization
probe and hand agitation
Sample: 0.1 g
Solvent: 4 mL (80% MeOH)
(homogenization) + 1 mL
(agitation)
Extraction time: 1 min
(homogenization) + 30 min
(agitation)
Temperature: RT
(homogenization) and 70 C
(agitation)
Technique: stirring
Solvent: 4 mL (80% MeOH)
(Homogenization) + 1 mL
(agitation)
(homogenization) + 30 min
(agitation)
Temperature: RT
(Homogenization) and 70 C
(agitation)
Proposed method and

reference method (modied
Murphy method)
Solvent: 4099.99% EtOH

Volume:sample ratio: 1:1 to
10:1 (mL g1 )
Temperature: 4090 C
Soybean our
Technique: Stirring
Sample: 0.5 g
Solvent: 25 mL
De: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MDi: malonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin, AGi: acetyl genistin, AGly: acetyl glycitin, MeOH:
methanol, EtOH: ethanol, CH3 CN: acetonitrile, RT: room temperature.
a
Tentatively identied by literature.
10
certain amount of water could optimize the total extraction. Extraction conditions were optimized for each soy sample. The amount of
water had a signicant effect of the amount of isoavone extracted
and varied with the food extracted. The amount of water optimized,
depending of the food matrix, ranged from 5 mL to 10 mL (isolate,
10 mL; tofu, 10 mL, soybeans, 7 mL, miso, 5 mL) using 2 g samples.
Also, the question of which extraction solvent is more efcient
is difcult to answer since it will depend of several factors such
as the technique, sample, amount of water, time, sample:solvent
ratios, temperature, etc. For example, while Murphy [99] observed
that 80% MeCN (with and without acid) was more efcient than 80%
MeOH (with and without acid), Barnes et al. [78] did not observe
signicant differences between 80% MeOH and acidied 80% MeCN
for the extraction of isoavones from toasted soy our toasted soy
our using a different solvent to sample ratio.
Later, Grifth and Collison [104] proposed an improved procedure for the extraction of isoavones from different soy samples
using 60% MeCN with 3% dimethylsulfoxide (DMSO) (v/v) and
compared this solvent with 80% MeOH. This procedure was also
compared with the modied Murphy [99] method used by Song
et al. [101]. 80% MeOH was less efcient than MeCN (with and
without acidication + DMSO) in extracting most isoavones and
differences between MeCN solvents (with and without acidication + DMSO) were smaller, with the primary difference in the
extraction efciency of more hydrophobic isoavones (AGi, Ge, De
and Gle).
Afterwards, different water proportions of the extraction solvent were tested and 60% MeCN proved to be the most efcient
solvent for two different soy protein samples (high and low in malonyl isoavones). It was also observed an improvement (0.710.6%)
in the extraction efciency of different isoavones from soy samples extracted with DMSO. The authors suggested that marginal
increase in isoavone content might be attributed to the lack of
acid or to the presence of small quantity of DMSO. It is clear that
more research is still needed to evaluate the inuence of DMSO
on extraction efciency of isoavones and examine the observations reported in this study. Another interesting result was the small
effect of extraction time and the observation that the vast majority
of isoavones were extracted in the rst 5 min of extraction. This
is strong evidence that the extraction time of similar methods can
be drastically reduced from 2 h and this parameter can be further
optimized.
Following the matter about the choice of the extraction solvent,
Murphy et al. [102], reviewed the extraction method and further
investigated MeCN, EtOH, ACE and MeOH in a 53% aqueous solution with and without acid addition using the same method and
concluded that MeCN was more efcient than the other solvents
and that MeOH was the least efcient solvent in extracting the
12 main isoavone forms in raw soy our, tofu, tempeh, texturized vegetable protein and soy germ. They also observed that the
different solvents have different abilities to extract the different
isoavone forms and that the food matrix conguration may have
an important impact on the extractability of the isoavone forms.
Depending of the sample, some solvents may underestimate individual isoavone content up to 35% and total isoavones up to 20%.
Another important remark was that addition of acid reduced the
extracted amount of some isoavones and increased the extraction
of others depending of the sample matrix. The authors suggested
that in order to simplify the extraction protocol, it is probably better
not to use acid in the extraction medium for these food matrices.
In fact, the addition of small quantities of acid to the extracting
solvent used by Murphy et al. [41,42,99,100] have been questioned
since no clear differences or systematic pattern for all foods or for
all isoavone forms have been demonstrated and as evidenced by
Grifth and Collison [104].
The initial purpose for the addition of small amount of acid was
to increase the extraction efciency and minimize coextractives
and give clean HPLC chromatograms. However, in the initial report
[99], non-acidied MeCN extracted lower amounts of coextractives
with similar efciency than acidied MeCN. Therefore, the use of
acidied MeCN seems not to make sense.
Further evidence is provided by Lin and Giusti [105], who evaluated the effects of solvent polarity and acidity on the extraction
efciency of isoavones from soybeans. In this report, acidied
solvents either extracted signicantly (p < 0.05) lower amounts
of isoavones or did not signicantly differ from solvents without acid. Non-acidied solvents were more efcient in extracting
malonyl isoavones. For glucosides isoavones, the acidication
showed a less signicant effect on Gi and Gly and no relevant effect
on Di. Also, no remarkable effect of acidication was found in the
extraction of AGi and aglycones (Ge and De).
The differences in the total isoavones obtained between acidied and non-acidied solvents mainly reected the differences in
malonyl isoavones. This may, in part, explain the results obtained
in the rst report of Murphy [99] regarding the use of acidied
solvents, since malonyl isoavones were not measured in this study.
Moreover, a signicant polarityacidity interaction was found
for aglycone extraction, which suggests that the effect of the acid
was not the same in the solvents with different polarities. Another
important observation was that acidication of the extraction solvent favored isoavone transformations during the extraction and
therefore should be avoided for quantication of intact isoavones
[105].
Regarding the extraction efciency of the solvents, results indicated that for all glucoside isoavones the solvent with higher
polarity (58% MeCN) either extracted signicantly higher amounts
or did not signicantly differ from the assayed solvents with lower
polarity (80% MeOH and 83% MeCN). The differences between 58%
MeCN (most polar) and 83% MeCN (least polar) were important in
terms of extraction efciency of individual and total isoavones.
However, differences between 58% MeCN and 80% MeOH or
between 80% MeOH and 83% MeCN were not always relevant.
On average, 58% MeCN extracted signicantly higher amounts
of malonyl glucosides than 80% MeOH and 83% MeCN. Recoveries of aglycones, Ge and De with 80% MeOH resulted signicantly
lower than those obtained with the other evaluated solvents. The
differences in measured isoavones between solvents with various
polarities reected the differences in malonyl glucosides, because
malonyl glucosides was the major form of isoavones in the soybeans and it was most affected by solvent polarity.
Therefore, solvents with relatively higher polarity and no acid
were more efcient in general for extracting isoavones. Among
the six examined solvents, 58% MeCN without acidication was
the best solvent for the extraction of isoavones from soybeans,
since it yielded the highest total amounts and best maintained the
intact structures. With regard to the two most widely used solvent systems, 80% MeOH had a higher extraction efciency and
better protection against chemical transformation than acidied
83% MeCN.
These results are in agreement with those reported by Rostagno
et al. [106] who compared different solvents for the extraction
of isoavones glucosides and MGi from soybeans. These authors
observed that when using pure solvents, low extraction efciency was obtained and that the maximum amount extracted was
obtained using solvents with 4060% of water. They also observed
that temperature has a great impact on the extraction efciency of
isoavones. Rostagno et al. [106] also reported that most isoavones
present in the sample (8090%) were extracted in the rst 10 min
of extraction at 60 C using 50% EtOH, corroborating similar observations reported by Grifth and Collison [104].
Another approach of solvent selection was given by Achouri et

al. [107]. These authors compared three solvents (80% MeCNHCl
0.1N, 80% MeOH and 80% EtOH) for the extraction of isoavones
from defatted soybean meal (DSM) and from soy protein isolate
(SPI). In the case of the DSM, the conclusion was that acidied 80%
MeCN is more efcient for the extraction of malonyl isoavones
and aglycones, while 80% MeOH is more efcient for the extraction of glucosides (using one extraction). In the case of the SPI, 80%
EtOH extracted the highest amount of aglycones, no signicant difference was observed between 80% EtOH and 80% MeOH for the
extraction of glucosides and that acidied 80% MeCN extracted the
highest amount of malonyl glucosides (using one extraction). However, 80% MeOH extracted the highest amount of total isoavones,
followed by 80% EtOH and by acidied 80% MeCN in this particular sample. These results indicate that the extraction efciency of
the solvent will depend of the sample from which the isoavones
are extracted. One of the most interesting remarks in this report
was that individual amount of isoavones extracted after the rst
extraction increased signicantly after 5 consecutive extractions
(42100% depending of the isoavone) in soy meal, and in soy
protein isolate (89153% depending of the isoavone). For the
different solvents used, the yield of total isoavones after 5 extractions (compared to only one extraction) increased between 65%
and 74% for the DSM sample, and increased by between 107% and
147% for ISP sample, depending of the solvent. The most important observation in this report was that no signicant difference
in terms of total isoavones was observed between the assayed
solvent after 5 sequential extractions in the DSM sample indicating that it is possible to achieve quantitative extraction with
any of the most commonly solvents used for isoavones extraction, given that conditions are optimized enough. Moreover, these
results strongly suggest that one time extraction of isoavone using
conventional methods markedly under-estimates the concentration of isoavones in these products.
However, sample characteristics are likely to play an important
role in the ability of a given solvent to extract isoavone from soybeans and soy foods. It is very interesting the observed variation
in the extraction yield of isoavones between DSM and SPI. For
the high protein sample (SPI), a unique extraction extracted only
41% of total isoavone compared to 58% of lower protein sample
(DSM) using MeCNHCl solvent. This difference was attributed to
stronger proteinpolyphenol interaction in the SPI sample since a
variety of interactions including hydrogen bonding, ionic and covalent binding, and mainly hydrophobic interactions are involved in
the formation of proteinpolyphenol complex [108]. These interactions are strongly inuenced by factors such as temperature, pH
and salt, which occur during acidic precipitation of soy proteins.
This outcome may also indicate that grinding and the resulting
particle size might, due to the effect in the matrix, can inuence
the ability of the different solvents to extract isoavones. The same
principle can be extended to freeze-drying, which more severely
affect sample matrix structures.
Another extraction method using 80% MeOH for the analysis of
isoavones from soybean our was later proposed by Tsai et al.
[103]. The proposed method was compared with a modied Murphy method (using different sample to solvent ratio). They observed
that, except De and Ge, contents of detected isoavones (Gi, Di, Gly,
MGi, MDi and MGly) extracted by the proposed method were higher
than those extracted by the modied Murphy method. These ndings imply that that several reports of isoavone distribution in
foods using the method by Murphy are underestimating isoavone
concentration.
On the other hand, Zhang et al. [20] evaluated several extraction conditions for the extraction and purication of isoavones
from soybeans. Extraction conditions included EtOH percentage
11
(4099.99%), solvent volume to sample ratio (1:1 to 10:1 mL g1 ),

temperature (4090 C) and extraction time (224 h). In this report,
the inuence of some extraction parameters was different than
those obtained by other authors. Pure EtOH extracted the highest
amount of isoavones, while in most studies is clear that a certain
amount of water (4060%) in the solvent is necessary to improve
extraction. Also, increasing solvent volume to sample ratio from 3:1
to 8:1 (mL g1 ) negatively affected yield. The objective of this study
however, is the key to understand the differences on the effect of the
sample amount observed by other authors. In this case, the objective was to extract the highest amount of aglycones and to obtain
a concentrated extract, not to quantify all chemical forms. Using
a higher amount of sample with lower amounts of solvent volume, it is logical that the concentration of isoavone on the extract
tends to increase although lower relative extraction efciency is
achieved.
Summing up, the differences among the extraction methods
reported in this review are most probably related with the amount
of water used in the extraction solvent, the sample matrix, the
extraction technique, sample to solvent ratio and more importantly,
the isoavone forms that were quantied. In some cases comparison of extraction solvents were carried out for only a few isoavones
present. In this context, it is important to note that some chemical forms are responsible for the greater part of the total amount
of isoavones present in soybeans and soy foods, especially some
malonyl and glucoside forms. Moreover, differences in analytical
methods and reporting of isomeric conversions can also contribute
signicantly to variation on the results found in the literature. In
some studies, total isoavone is expressed as the sum of all 12 isomers. In other studies, only aglycone and/or conjugated forms are
tested and expressed. Furthermore, in other studies isoavones are
hydrolyzed to their aglycone forms or the amount is normalized by
molecular weight to the aglycone forms. Also, some methods were
developed before the malonyl glucoside isoavones were identied [38,39] and therefore the results needed to be revised for the
extraction of all 12 isoavone forms. More importantly, the effect
of extraction conditions on stability was not considered in many
cases.
In general terms, the choice of the most appropriate solvent will
depend of the isoavone in highest amount present in the sample, since the most effective solvent for this particular isoavone
will strongly inuence the total amount extracted. For comparison purposes it is important to evaluate different solvents without
achieving quantitative recoveries otherwise it will be impossible
to determine the magnitude of effect of the solvents. However, the
recent trend is to avoid toxic and use environmental friendly solvents such as EtOH. EtOH can be highly effective for the extraction
of isoavone from soy samples with the advantage of lower cost,
lower toxicity and environmental compatibility.
It also appears clear that to obtain quantitative extraction for the
analysis of the isoavone content of foods is necessary to adjust
extraction conditions for each sample and some research is still
needed to optimize other extraction variables, especially sample to
solvent ratio and extraction time.
One of the important conclusions when reviewing information
available is that it is possible to achieve quantitative extraction using most commonly used solvents and that it very likely
that sequential extractions are required, as previously mentioned.
Finally, more research is needed to evaluate and explain the inuence of the sample, since it may be the answer to achieve standard
methods for the extraction and analysis of isoavones in foods.
5.1.1.1. Stability during extraction using conventional techniques.
Apart of optimizing extraction variables such as solvent, sample
amount, temperature and duration, the assessment of stability
12
during extraction is essential. Frequently, authors tend to overextend extraction duration in order to achieve higher extraction
yields. This strategy, however, may cause not only degradation of
some chemical forms, but also the generation of other isoavones
forms and isomers that can drastically modify isoavone prole of
the sample and inuence the results obtained. Thus, although long
extraction times have been extensively used for the extraction of
isoavone from soy and other matrixes, there are still several issues
that should be addressed such as the stability during extraction.
Extraction of isoavones from foods or dietary supplements
is a critical process since isoavone prole can be altered during sample preparation since mild heat and acid are frequently
involved in the extraction, which could cause degradation of malonyl isoavones and the hydrolysis of glucosides. Therefore, when
choosing the extraction conditions it is important not only consider extraction efciency, but also avoid, or at least minimize, the
articial transformations. Thus, temperature conditions during the
extraction procedures as well as extraction duration have to be
carefully adjusted because of possible degradation of the glucoside derivatives. Also, stability may be related to the solvent used,
specially acidied solvents.
One of the earlier observations of the inuence of the extraction temperature on the isoavone prole was reported by Kudou
et al. [39]. They observed that malonyl isoavone glucosides in
70% alcohol extracts from both soybean hypocotyls and cotyledons
decreased signicantly as their respective glucosides increased
when the samples were extracted at 80 C instead of room temperature.
The effects of extraction temperature on isoavone prole were
later conrmed by Barnes et al. [78]. They observed that extractions
performed at 60 C caused heat induced de-esterifying reaction of
malonyl and acetyl glucosides to their respective glucosides and
that increasing temperature to 80 C led to higher conversion rate.
Moreover, the changes on isoavones prole were not only due
to temperature variations, but also time dependent. Even at room
temperature malonyl glucosides were gradually converted to their
respective glucosides. The conversion rate at room temperature
was later reported to be between 0.2 mol% and 0.3 mol% per hour
[42]. Obviously, extraction methods using long extraction times can
signicantly underestimate malonyl glucoside concentration and
overestimate glucoside concentration.
Coward et al. [71] evaluated the effect of the temperature on the
extraction of isoavones from soy foods. Isoavone -glucosides
conjugates were extracted with 80% MeOH from soybeans at room
temperature, at 4 C and at 80 C, for 272 h by tumbling or shaking.
Quantitative and reproducible recovery of the isoavone glucosides
was achieved after 2 h. Extraction at 4 C gave the highest concentration of malonyl glucosides and the lowest concentration of
-glucosides conjugates. Extraction at 80 C caused extensive conversion of the malonyl glucosides conjugates to the -glucoside
conjugates but not to the acetyl conjugates or aglycones. Although
the composition of the individual -glucosides was drastically
altered by temperature, the total amount of isoavones extracted
was constant.
On another study, Franke et al. [93] evaluated the stability
of De, Ge, coumestrol, formononetin, biochanin A and avone
under reuxing for 4 h using acidied 77% EtOH (2.0 M HCl) and
observed that only avone was entirely stable. Therefore, it is clear
that reuxing is not recommendable for extraction of isoavones
from soybeans and soy foods, since it can cause losses, even if
hydrolytic methods are used. However, this may be related to the
use of acidied solvent as reported by Lin and Giusti [105], who
later observed the transformation of -glucosides to their corresponding aglycones and transformation of acetylglucosides to
their corresponding -glucosides when subjected to extraction by
stirring for 2 h at room temperature. Another important remark

was that acidication of the extraction solvent favored isoavone
transformations during the extraction and should be avoided for
quantication of intact isoavones.
Therefore, when evaluating an extraction method it is of crucial importance to know the stability of target compounds, in order
to maintain the isoavone prole in the sample, unless hydrolytic
methods are used (see Section 4). Submitting an extract obtained
with optimal conditions to the extraction protocol and comparing concentrations is a simple way to perform such stability tests
[109,110]. It cannot guarantee that target compounds are entirely
stable since other sample matrix components may inuence stability, but may give clues about the possible degradation under
extraction conditions. The use of extracts is preferable to the use of
standards since extracts contain other components and are more
close to real samples. Another method is to control concentration
of malonyl isoavones trying to identify degradation patterns or
use hydrolytic methods, quantifying aglycone equivalents. The use
of an internal standard may also prove useful in this case.
The most recent trend regarding stability during extraction is to
use of -glucosidase inhibitors. Toebes et al. [111] identied Tris as
a suitable -glucosidase inhibitor in red clover extracts, which was
optimized at 350 mM in 80% EtOH at pH 7.2. Extractions using Tris
yielded much higher amounts (1324 times) of malonyl isoavones
as opposed to extractions without Tris. Although it was evaluated
for the extraction of isoavones from red clover, the same principle may be applicable for the extraction of soy isoavones. In
this case, however, concentration of Tris might need adjustment
and further investigation, but unveils a strategy to avoid degradation and, therefore, increase the reliability of results obtained in the
future. Other possible candidates for this role are HgCl2 , AgNO3 and
d-glucono--lactone, which have been reported to inhibit soybean
-glucosidase, being the later the most potent inhibitor [112].
Although important advances have been made regarding the
stability of isoavones during extraction using conventional techniques, it is clear that more studies are necessary, especially with
the aim of avoiding degradation in order to provide reliable information about the concentration of these compounds in foods. Also,
sample and solvent characteristics have to be further examined in
detail as well as other factors such as temperature and extraction
technique.
5.1.2. Modern extraction techniques and methods
The development and application of modern samplepreparation techniques with signicant advantages over conventional methods (e.g. reduction in extraction time, organic solvent
consumption and in sample degradation, elimination of additional
sample clean-up and concentration steps before chromatographic
analysis, improvement in extraction efciency, selectivity, and/or
kinetics, ease of automation, etc.) for the extraction and determination of isoavones from soybeans and derived foods is playing an
important role in the overall effort of ensuring and providing high
quality data for researches worldwide.
With this in mind, newer extraction methods have been developed using modern extraction techniques, including supercritical
uid extraction, ultrasound-assisted extraction (UAE), pressurized
liquid extraction (PLE), microwave-assisted extraction and solid
phase extraction.
When selecting the appropriate solvent for the extraction of
isoavones using conventional extraction techniques, solubility is
one of the most important factors. However, the selection of an
appropriate solvent using modern extraction techniques is much
more complex, since it will depend of other factors besides of the
solubility of target compounds, such as the ability of the solvent
to absorb microwave energy (MAE), how it propagates ultrasonic
waves (UAE) and the physicalchemical changes in the solvent that

take place under elevated temperature and pressure, which will
also affect solubility of target compounds (PLE/SFE).
5.1.2.1. Ultrasound-assisted extraction. The enhancement of extraction efciency of organic compounds by ultrasound is attributed to
the phenomenon of cavitation produced in the solvent by the passage of an ultrasonic wave. Cavitation bubbles are produced and
compressed during the application of ultrasounds. The increase in
the pressure and temperature caused by the compression leads to
the collapse of the bubble, resulting on a shock wave that passes
through the solvent enhancing the mixing. Ultrasound also exerts
a mechanical effect. When a bubble collapses near a solid surface
it occurs asymmetrically and generates high-speed jets of solvent
towards the cell walls, therefore increasing the solvent penetration
into the cell and increasing the contact surface area between solid
and liquid phase. This effect coupled with the enhanced mass transfer and signicant disruption of cells, via cavitation bubble collapse,
increases the release of intracellular product into the bulk medium.
The use of higher temperatures in UAE can increase the efciency
of the extraction process due to the increase in the number of cavitation bubbles formed. Several extraction parameters, similar to
conventional extraction methods, can inuence the extraction of
organic compounds using ultrasounds, such as polarity and amount
of the solvent, the mass and kind of sample and extraction time
among others. Also, parameters regarding the ultrasound source
such as frequency and intensity as well as the number of pulses
applied can have great impact on extraction dynamics [113117].
Ultrasound-assisted extraction has been used in several occasions to extract isoavones from soybeans, soy foods and from
different matrixes, such as Peanuts, Trifolium pretense, Puerariae radix, Pueraria lobata, Radix astragali and Glycyrrhizae radix
[80,82,106,118125]. However, optimization of UAE based methods has not been conducted with a few exceptions. An overview
of the developed methods using ultrasounds for the extraction of
soy isoavones and evaluated parameters is presented in Table 2.
One of the rst methods where extraction conditions were
systematically assessed to achieve quantitative extractions of soy
isoavones was published by Rostagno et al. [106]. For the method
development, several extraction parameters were studied including solvent, extraction temperature, sample amount and extraction
time. The most important parameters affecting the extraction efciency were the extraction solvent (and the amount of water),
extraction temperature and extraction time. The extraction efciency was improved by using ultrasounds but was dependent of
the solvent employed. 50% EtOH, 50% MeOH and 40% MeCN were
the solvent that extracted the highest amount of total isoavones
with similar extraction efciency. The best extracting solvent for
each isoavone form depended of the chemical form itself. For
all chemical forms best extraction efciency was achieved using
solvents with 4060% of water.
Extraction temperature had a great impact on the extraction efciency while using higher temperature signicantly increased the
amount of all tested isoavones. In general, the method was found
to be fast and reliable achieving quantitative extractions in 20 min.
To be sure that quantitative recovery was achieved; results were
compared with 5 sequential extractions with no signicant difference. Most isoavones (8090%) occurring in the soy our sample
were extracted in 10 min; corroborating the results obtained by
Grifth and Collison [104] (see Section 5.1.1). Extending the extraction length to 30 min decreased the yield of some isoavones. Also,
no signicant difference was observed between ultrasonic probe
and ultrasonic bath and therefore, it can be used as an alternative
with the advantage of allowing the extraction of multiple samples.
The method developed by Rostagno et al. [106] has been used, with
13
and without modications, for routine analysis, to obtain isoavone

extracts for other studies and as reference method for comparison
of other extraction methods [80,82,109,110,119,126138].
Regarding the extraction solvent, Achouri et al. [107] compared
three solvents (80% MeCN + HCl 0.1N, 80% MeOH, 80% EtOH) for
the ultrasound-assisted extraction of isoavones from different
matrixes (defatted soybean meal and from soy protein isolate)
and observed that 80% MeOH and 80% EtOH extracted the highest
amount of isoavones from both samples. They also observed that
sonication for 15 min extracted as much as the total of 5 sequential
extractions (with ordinary shaking for a total of 10 h), except for
acidied MeCN. This is an important observation, since acidied
MeCN is one of the most used solvent with conventional extraction
techniques and points that it is not recommendable to use this solvent when using ultrasounds, since it can seriously underestimate
isoavone content of foods. It was also observed that extending the
time of sonication from 15 min to 30 and 60 min, did not increase
the total amount of isoavone extracted, and in some cases the
total amount decreased, corroborating the observations made by
Rostagno et al. [106].
More recently, Bajer et al. [129] compared pure MeOH, MeCN
and ACE for the extraction of De and Ge from soy our. MeCN
gave the highest yields and was further studied adding different
amounts of water (050%) and 60% MeCN gave the best results.
Temperature was also evaluated between 25 C and 80 C as well
as extraction time between 10 min and 50 min. Highest amounts
of isoavones were obtained at 50 C for 40 min using the ultrasonic bath. Also, using an ultrasonic homogenizer pulse generator
was evaluated in the range of 4598 W (100%) the use of ultrasonic
pulses during extraction and the extraction time in the range of
1050 min. Best extraction yields were obtained using 60% of ultrasonic amplitude for 30 min. These results were obtained at room
temperature. In this report, unfortunately, information of the inuence of studied extraction conditions and their respective data was
not given, only a few isoavones were studied and was limited in
terms of the types of samples evaluated.
The inuence of ultrasound on the solidliquid extraction process as regards yields or selectivity is very difcult to predict
because of the interaction of many factors, either relative to the
phase system (solid, liquid/solute) or to the ultrasonic reactor itself.
The differences observed on the amount of water used in the solvent by the different reports may be related with the type of sample
used and its characteristics. It is very likely that the amount of water
needed to achieve maximum extraction efciency might need some
adjustment depending of the sample type, as reported by Murphy
et al. [42] using conventional stirring.
Other factors may be inuencing the extraction dynamics, since
UAE is affected by the ultrasonic wave distribution inside the
extractor. Maximum ultrasound power is obtained at the vicinity of the radiating surface of the ultrasonic source and an abrupt
decrease of the ultrasonic intensity increases as the distance from
the radiating surface increases. Furthermore, the presence of solid
particles can affect the ultrasonic intensity prole, which can be
are attenuated depending of nature of the sample such as hardness,
compactness, particle size and solute distribution [130].
Also, the inuence of other important extraction variables such
as frequency, intensity and the use of ultrasonic pulses have not
been extensively studied in detail and for all isoavones and therefore future investigations should focus on these issues as well as on
the inuence of the sample on the extraction. In general, UAE seems
a potent technique for the extraction of isoavones from soybeans
and soy foods. This technique can achieve high extraction yields
in less than 30 min from different sample types using the commonly used solvent in conventional methods. It clear though, that
high temperatures can be used since extractions are short and that
14
Table 2
Developed methods using ultrasounds for the extraction of soy isoavones and evaluated parameters.
Sample used for evaluation
of the method
Defatted soybean meal and

soy protein
Isoavones
Di, Gi, Gly and MGi
Di, Gi, Gly, De, Ge,

Gle, MDia , MGia and
MGlya
Fixed extraction
conditions
Solvent: 25 mL
Vibration
amplitude: 100%
Sample: 2 g
Solvent: 10 mL
Temperature: 22 C
Ultrasound source:
ultrasonic bath
Solvent:
EtOH (3070%)
MeOH (3070%)
CH3 CN (3070%)
Sample amount: 0.50.1 g
Ultrasound source: ultrasonic probe and
ultrasonic bath
Solvent:
80% EtOH
80% MeOH
Selected conditions
Reference
50% EtOH, 60 C, 0.1 g, 20 min
[106]
80% MeOH and 80% EtOH,

15 min
[107]
80% CH3 CN (0.1N HCl)

Soy our
De and Ge
Sample: 1 g
(ultrasonic bath)
and 2 g (ultrasonic
homogenizer)
Solvent: 25 mL
(ultrasonic bath)
and 45 mL
(ultrasonic
homogenizer)
Temperature: RT
(ultrasonic
homogenizer)
Solvent:
EtOH
MeOH
CH3 CN (50100%)
60% CH3 CN
Temperature: 2580 C (ultrasonic bath)
Ultrasonic bath: 50 C,
40 min
Ultrasonic homogenizer:
30 min, and 60% vibration
amplitude, pulse generator
(not specied)
[129]
Ultrasound source: ultrasonic bath and

ultrasonic homogenizer
Pulse generator: 4598 W
Vibration amplitude: range not specied
De: daidzein, Ge: genistein, Gle: glycitein, Di: daidzin, Gi: genistin, Gly: glycitin, MDi: malonyl daidzin, MGi: malonyl genistin, MGly: malonyl glycitin, ADi: acetyl daidzin,
AGi: acetyl genistin, AGly: acetyl glycitin, MeOH: methanol, EtOH: ethanol, CH3 CN: acetonitrile, RT: room temperature.
a
Tentatively identied by literature.
intermediate to high amounts of water in the extraction solvent

(4080%) are needed to efciently extract isoavones.
The inuence of ultrasounds on isoavone distribution during extraction should not be neglected. The same principles of
isoavone stability during extraction using conventional techniques apply when using ultrasounds. However, there are other
factors which can affect stability of these compounds such as the
production of radicals from the ultrasound dissociation of water.
In the presence of these high energy species, oxidative reactions
can take place simultaneously with the extraction reactions when
water is higher than 50% [106]. This is particularly important, since
as previously mentioned intermediate to high amounts of water
in the extraction solvent (4080%) are needed to efciently extract
isoavones.
Rostagno et al. [106], for example, observed a reduction of the
extraction efciency common to solvents with high amounts of
water (>60%) which was attributed to an increased production of
radicals from the ultrasound dissociation of water. In such report
slightly lower yields of total isoavones were obtained using extractions of 30 min with 50% EtOH at 60 C than those obtained with
extractions of 20 min. Similar results were obtained by Achouri et
al. [107], who observed that, in some cases, the total amount of
isoavones decreased if extraction were extended from 15 min to 30
and 60 min. In view of this evidence it seems advisable to use short
extractions rather than long extractions when using ultrasounds.
Stability of isoavones during UAE has not been apparently studied to the moment. With the available evidence that relatively short
extraction times can affect isoavone prole and content, assessment of the inuence of ultrasounds on isoavone degradation is
of one of the most urging needs in future research in this eld. The
effect of extraction solvent, temperature, ultrasound intensity and
frequency on stability of soy isoavones during UAE and the search
of effective ways to avoid degradation need to be further examined
in detail in future investigations.
5.1.2.2. Pressurized liquid extraction. Pressurized liquid extraction
is a procedure that combines elevated temperature (50200 C)
and pressure (100140 atm) with liquid solvents (without their
critical point being reached) to achieve fast and efcient extraction of the analytes from the solid and semi-solid samples matrix.
This technique has received different names, such as accelerated
solvent extraction (ASE), pressurized liquid extraction (PLE) and
pressurized solvent extraction (PSE). When water is employed as
the extraction solvent, the authors tend to use a different name,
such as superheated water extraction (SWE), to highlight the use
of this environmental-friendly solvent.
For rapid and efcient extraction of analytes from solid matrices, extraction temperature is an important experimental factor.
Elevated temperatures can lead to signicant improvements in
extraction efciency, since it may increase solubility of target
compounds, diffusion rates and mass transfer of analytes to the solvent. Moreover, temperature can dramatically modify the relative
permittivity of the extracting solvent, increasing selectivity. High
pressure allows maintaining the solvent in a liquid state at high
temperature and may increase the penetration of the solvent in the
sample matrix. Extractions performed under elevated temperature
and pressure results in adequate kinetics of dissolution processes
and favors desorption of analytes from the surface and active sites
of solid sample matrices [67,113,114,131,132].
PLE has been used in several occasions to extract isoavones
from soybeans, soy foods and other different matrixes such as Radix
puerariae, Matricaria recutita, Rosmarinus ofcinalis, Foeniculum vulgare and Agrimonia eupatoria L. [109,121,129,133140]. An overview
of the developed methods using pressurized liquids for the extraction of soy isoavones and evaluated parameters is presented in
Table 3.
The same principles of isoavone stability during extraction
using conventional techniques also apply when using PLE. The use
of high temperatures can strongly affect isoavone content and
prole as previously discussed in Section 3. In the case of PLE
however, temperatures used are much higher than those used in
conventional methods and thus it can expected that the extend of
degradation and transformations taking place during extraction are
much more important.
Rostagno et al. [109] evaluated the inuence of several extraction parameters, such as solvent, temperature, pressure, sample
size, static extraction cycle length and number of static extraction cycles in order to optimize extraction conditions to achieve
quantitative recoveries of isoavone from freeze-dried soybeans.
They observed that using EtOH/water mixtures, extraction efciency increased when increasing the water percentage in the
extraction solvent from 0% to 30%, and that higher amount of
water in the extraction solvent resulted in a lower extraction
efciency. Similar results were obtained for MeOH/water mixtures, and the highest extraction efciency was achieved using 60%
MeOH. Water extracted the lowest amount of isoavones between
assayed solvents. They also reported that increasing the extraction temperature from 60 C to 150 C increased the yield of most
isoavones (except the malonyl forms) and identied a degradation pattern. The increase in the extraction temperature from 60 C
to 100 C increased the total amount of isoavones extracted in
approximately 20%. The increase of the yield of isoavone glucosides with the increase of temperature between 100 C and
150 C was very pronounced (approximately 30%) while the yield
of malonyl isoavones decreased (approximately 50%), when it
was expected to follow the same trend as the glucoside forms and
increase.
Searching for answers, a stability evaluation of extraction conditions was performed which conrmed that degradation starts
above 100 C for the malonyl forms and above 150 C for the
isoavone glucosides. Above 100 C, with the decrease of MGi, a
correspondent increase in Gi concentration was observed. Concentration of other glucosides also increased at this temperature.
Aglycone levels remained constant below 150 C indicating that
degradation of glucosides was not taking place below this temperature. Above 150 C, aglycone levels showed a small increase
with the decrease in their respective glucoside levels, indicating the
conversion between these chemical forms. The stability study conrmed the observations made during the extraction temperature
optimization, indicating that 100 C is the maximum temperature
for PLE of isoavones. It was also reported that the increase of pressure from 100 atm to 200 atm did not have a signicant impact on
the extraction of isoavones from freeze-dried soybeans, and that
reducing sample size (from 0.5 g to 0.05 g) increased the yield of
isoavones in approximately 13%. However, relative standard devi-
15
ation increased proportionally. The extension of the three static

extraction cycle used from 5 min to 7 min increased the extraction
yield in approximately 10% and no signicant effect was observed
between 7 min and 10 min. To ensure that quantitative extraction
was obtained, the authors performed four re-extractions of the
sample achieving similar recoveries.
With the evidence provided by this report, it is clear that caution
should be used when increasing the extraction temperature and
that more research is needed to evaluate the stability of isoavones
during PLE. Among the main factors that should be studied in detail
in future researches are the inuence of the sample, solvent and the
duration of the procedure.
An interesting method for the extraction of isoavones from soybean foods was developed by Klejdus et al. [135] using PLE with
modied extraction cell content. The modication in the extraction cell content was made by using 5 mL of a commercial matrix
(SPE-edTM matrix). For the development of the method, similar
extraction parameters were evaluated, like solvents, number of
extraction cycles, sample amount, pressure and temperature. Additionally, another innovation of the method was the evaluation
of the effect of sonication time before PLE. The extraction yield
dramatically increased by using sonication before PLE extraction
(performed with 90% MeOH). The amounts of extracted individual isoavones rapidly increased with the sonication time up to
1 min, and using longer sonication times the increase was lowered
and it was nearly constant after 5 min. However, extraction yield of
aglycone (De and Ge) continuously increased with increasing sonication time until 5 min. The increase in the extraction efciency
was attributed to the disruption of cell walls by ultrasonic waves.
Regarding extraction cycles (performed with 90% MeOH), highest
isoavone concentrations in the extracts were obtained after three
extraction cycles. However, differences in the yield between two
and three cycles were only about 5%.
Most often used solvents (i.e. MeCN, EtOH and MeOH (50 or
6090% in water)), were evaluated for the extraction under PLE.
The extraction yields obtained for the extraction of Di and Gi with
MeCN were about 6080% (depending of the water percentage) of
the yields of the extraction with MeOH (90%). The extraction efciency rapidly decreased with the increasing content of MeCN in the
case of Di. Extraction yields between 60% and 75% of the amount
extracted with MeOH (90%) were obtained using EtOH with different amounts of water (6090%). Highest yields of both isoavones
were obtained using 90% MeOH, and linear decrease of extraction yields were obtained with decreasing content of MeOH in the
extraction agent.
Using different amounts of sample, the authors observed
decreasing yields with the increasing amounts of sample, obtaining similar results as those obtained by Rostagno et al. [109]. This
nding was attributed to the thicker layer of sample in the extraction cell. In such study, the inuence of pressure was also evaluated.
The decrease in the sample size from 0.5 to 0.1 increased approximately 40% the amount of Di and Gi extracted. With the increase of
pressure from 13 kPa to 14 kPa of pressure the amount extracted
of both isoavones increased, and no signicant difference was
observed between 14 kPa and 15 kPa. Increasing extraction temperature from 70 C to 110 C produced an increase of the extraction
yield of Di and Gi (15%), and a much more dramatic increase was
observed between 110 C and 145 C (60%). The authors claimed that
temperature of about 145 C was most suitable for obtaining maximal efciency. The optimized method was used for the analysis of
several isoavones different soy samples.
However, the greatest concern regarding the proposed method
is that the malonyl forms were not measured and stability was
not evaluated. Using as reference the stability study made by Rostagno et al. [109] where most malonyl isoavones are not stable
16
Table 3
Developed methods using pressurized liquids for the extraction of soy isoavones and evaluated parameters.
Sample used for
evaluation of the
method
Soy bits
Isoavones
Di, Gi, Gly and

MGi
Di, Gi, De and

Ge
Extraction cell: 11 mL
Inert material: sea sand
Solvent:
EtOH (3080%)
MeOH (3080%)
Water
Sample amount: 0.2 g

Cell content: 5 mL of a commercial
matrix (SPE-edTM matrix)
Static cycle length: 5 min
Selected conditions
Reference
0.1 g, 100 C, 70% EtOH,

3 7 min static cycles
(22 mL)
[109]
1 min sonication time,

0.1 g, 90% MeOH,
14 kPa, 2 static cycles
(20 mL)
[135]
70% EtOH + 5% DMSO
[134]
CH3 CN, 2 static cycles,

of 15 min
[129]
110 C, 641 psig, 2.3 h
[139]
80% EtOH, 383 K,

551 kPa, 25 mL/min,
80 g
[140]
Pressure: 100200 atm

Number of static cycles: 13 (7 min)
and 12 (10 min)
Solvent:
CH3 CN
EtOH (5090%)
MeOH (5090%)
Pressure: 1315 kPa

Sonication time: 15 min
Number of static cycles: 13
Soybeans
Soybean our
Di, Gi, Gly, De,

Ge, Gle, MDi,
MGi, MGly, ADi,
AGi and AGly
De and Ge

Inert material: Ottawa sand
Pressure: 1000 psi
Solvent:
58% CH3 CN, 58% CH3 CN + 5% DMSO
70% EtOH, 70% EtOH + 5% DMSO
90% MeOH
Temperature: 100 C
Number of static cycles: 3
Water
95% Genapol
Sample amount: 2 g
Temperature: 100 C
Solvent: MeOH, ACE, CH3 CN

Pressure: 515 MPa
Inert material: quartz wool/glass

beds
Number of static cycles: n.e.

Extraction time: n.e.
Defatted soybean
akes
Defatted soybean
akes
Di, Gi, Gly, De

and Ge
Di, Gi, Gly, De

and Ge
Sample amount: 180 g

Extraction cell: 2 L
Temperature: 60130 C
Pressure: 300735 psig
Solvent: 1800 mL of water
Temperature: 333393 K
Pressure: 4134410 kPa
Solvent ow rate: 1025 mL/min
Solvent: EtOH:water ratio (095%)

AGi: acetyl genistin, AGly: acetyl glycitin, MeOH: methanol, EtOH: ethanol, CH3 CN: acetonitrile, n.e: not specied.
under PLE above 100 C (using 70% EtOH), and a similar dramatic
increase on the yield of glucosides was observed at 150 C, part
of the effect of increasing the temperature in the increase of the
extraction yield of glucosides may be attributed to degradation of
malonyl isoavones. Corroborating evidence is that yield of glucoside decreased when extractions were performed above 145 C, as
reported by Rostagno et al. [109]. Therefore, the proposed method
may not be able to extract all isoavones and more importantly,
without changing the isoavone prole of the sample. The same
method with slight modication (i.e. sonication time of 5 min
instead of 1 min) was used for evaluation of isoavone aglycone
and glucoside distribution in soy plants and soybeans by Klejdus et
al. [136].
Also, Klejdus et al. [137] improved the previous method and used
a two phase PLE extraction program combined with UAE to extract
isoavones from soy bits. In the rst PLE phase, the sample was
extracted with 2 cycles of 5 min each with hexane at 145 C using
145 bar of pressure, followed by a second phase of 2 cycles of 5 min
each with 90% MeOH at 145 C using 145 bar of pressure. This is an
interesting approach since it allows cleaning the sample and performing the extraction of target compounds without manipulation
of sample, avoiding the associated errors. However, the same stability issues of the original method [136] persist in the improved
method.
Later, Luthria et al. [134] using the method developed by
Rostagno et al. [109], compared several extraction solvents (58%
MeCN, 70% EtOH, 90% MeOH, Water and 95% Genapol) and
evaluated the inuence of the addition of 5% DMSO to the
extraction solvent for the extraction of isoavones from soybeans. They observed great differences between assayed solvent.
Both, the total isoavone content and the isoavone HPLC prole
varied signicantly with different extraction solvents, achieving
highest total isoavone recoveries from soybean samples with
DMSO:EtOH:water. 58% ACN extracted only 30.5% of the isoavones
extracted with DMSO:EtOH:water. With the addition of DMSO to
58% ACN improved extraction to 52.3%. The addition of DMSO to
70% EtOH also improved extraction efciency, while 90% MeOH
achieved intermediate yields (83.7%). Very low efciency was
obtained with genapol or water (18.2% and 13.7%, respectively).
However, since extraction conditions used were optimized by
Rostagno et al. [109] for 70% EtOH, it was expected that the maximum efciency was obtained using DMSO:EtOH:water (5:70:25)
and EtOH:water (70:30). On the other hand, useful information
is provided by the improvement of extraction by DMSO. A possible explanation to the improvement of the extraction efciency
was attributed to the solubility of isoavones in DMSO reported
by SigmaAldrich web site (http://www.sigma-aldrich.com). The
authors also observed important differences among assayed solvents. 90% MeOH extracted the highest amount of glucosides (Gi,
Di and Gly) and while for the other nine isoavones the best
solvent was DMSO:EtOH:water (5:70:25). This may explain the
results obtained by Klejdus et al. [135], achieving best yields
with MeOH than with EtOH, since only glucosides and aglycones
were quantied. An interesting observation was the detection
of all 12 isoavones by only two extraction solvent mixtures
(DMSO:EtOH:water (5:70:25) and (DMSO:MeCN:water (5:70:25)).
De and MGly were not extracted at detectable levels by the other
solvents. Similarly, Klejdus et al. [136] extracted only trace amounts
of De (1.2% of total isoavones) and did not detect MGly when using
90% MeOH.
In contrast, Bajer et al. [129] observed that out of three solvents
tested (MeOH, MeCN and ACE) MeCN gave the highest yields at
100 C. However, extraction was evaluated for some aglycones (De
and Ge only) and the use of a certain amount of water in the extraction solvent was very likely to have inuenced the results obtained.
After the pressure was optimized in the range of 515 MPa, the
number of cycles and extraction time were also optimized using
MeCN. Unfortunately, data regarding the method optimization and
of the inuence of the extracting variables were not given.
With a different optimization strategy, Li-Hsun et al. [139] used a
steepest ascent design to examine the effect of several independent
variables (temperature, pressure and duration) on the extraction of
isoavones from defatted soybean akes by superheated water at
elevated pressures. They observed that temperature has a greater
impact than pressure and then time, in the extraction of isoavones
using water. The experimental design revealed that the optimal
condition for the extraction of isoavones was 110 C and 641 psig
(4520 kPa) for 2.3 h using 180 g of sample and 1800 mL of water.
When extractions were carried out at higher or lower temperature,
or with lower pressure, the total amount of isoavones decreased.
The authors concluded that the decreasing dielectric constant ()
of water at elevated temperature and pressure might play an
important role for the enhanced extraction of isoavones. This
is indication that the dramatic changes in the physicalchemical
properties of water, especially in its dielectric constant, at elevated
temperatures and pressures enhance its usefulness as extraction
solvent.
The low extraction efciency of water observed by Rostagno et
al. [109] when compared to the above results can be explained by
the use of a lower temperature (60 C), which is not high enough to
change the dielectric constant of water and increase its effective-
17
ness. An important remark is that not all isoavone chemical forms

were quantied. Also, the large sample amount and solvent volume
and the inability to extract some isoavones limit its usefulness as
an analytical method. However, the reported results provides an
important evidence that isoavones can be extracted using pressurized water if conditions are optimized enough which could be
exploited in future investigations.
Following the same direction, Chang and Chang [140] examined
the effect of pressure, temperature, solvent ow rate, EtOH:water
ratio, and the feed loading on the PLE of isoavones from defatted
soybean akes using water as solvent. Initially, the effect of solvent
ow and extraction temperature were evaluated and was observed
that using hot pressurized water, increasing the solvent ow rate
increased the extraction efciency as the extraction time increased
from 40 min to 200 min. The effect of the increasing temperature
was noticeable between 333 K and 383 K but not between 383 K and
393 K (all extraction were performed at 4410 kPa) independently of
the solvent ow rate (5 and 10 mL/min). These results are similar
to those obtained by Li-Hsun et al. [139].
Using pure water or EtOH (at 383 K and 4410 kPa) the later
procedure extracted more than 50% more isoavones than water.
Decreasing feed loading from 480 g to 180 g increased isoavone
extraction efciency in approximately 20% after 6 h of extraction,
being in agreement with the trends observed by Rostagno et al.
[109] and Klejdus et al. [135]. Pressure, however, did not significantly affect the PLE using EtOH with 360 g of feed loading as
reported by Rostagno et al. [109]. In contrast, solvent ow have had
an important effect on extraction efciency, and increasing ow
rate from 10 mL/min to 25 mL/min increased the total amount of
isoavones extracted in approximately 15% after 360 min.
The optimization of EtOH:water ratio, feed loading, pressure and
solvent ow rate on the recovery of isoavones of the PLE at 383 K
and 2.3 h was made by means of a four factor Taguchi experimental
design. EtOH:water ratio and ow rate were the parameters with
the highest inuence on the extraction efciency, while small differences were observed while evaluating feed loading and pressure.
In general, the higher the EtOH:water ratio and the ow rate, the
higher was the recovery.
Among the variables affecting PLE, the nature of the extraction solvent and temperature generally have profound effects on
the PLE process. When dealing with pressurized solvents, temperature will have different impact depending of the solvent used
since physicalchemical properties of each solvent are different.
Therefore, the best extraction conditions will depend of the solvent. The dramatically changes in the physicalchemical properties
of water, especially in its dielectric constant () at elevated temperatures and pressures enhance its usefulness as an extraction
solvent. The dielectric constant is a key parameter in determining solutesolvent interactions, and increasing the temperature
under moderate pressure can signicantly decrease this constant.
At ambient pressure and temperature, water is a polar solvent with
a dielectric constant ( = 78) but at 300 C and P = 23 MPa this value
decreases to 21, which is similar to the value of EtOH ( = 24 at 25 C)
or acetone ( = 20.7 at 25 C). This means that at elevated temperatures and moderate pressures the polarity of solvent can be reduced
considerably and water can be used instead of another organic solvent to extract medium-low polarity compounds, or reduce the
amount of the organic solvent used to achieve effective extraction
rates [67]. Due to the advantages of lower cost, environmental compatibility and toxicity, water can be used as extracting solvent if
high efciency is not required. However, in view of the available
literature, pure water, even at elevated temperature and moderate
pressure, is not as efcient as other solvents for the extraction of
isoavones, despite that the addition of certain amount of water to
the organic solvent is necessary to improve extraction efciency.
18
In general, it is clear that at higher temperatures extraction efciency tend to increase, independently of the solvent employed and
pressure is usually a minor variable (except when using water as
solvent) for the resulting efciency and that it is only required to
maintain the solvent in the liquid phase. Another important aspect
of PLE is the stability of isoavones under extraction conditions.
Since some isoavone are very sensitive to temperature and PLE
is performed at elevated temperatures, stability may be the limiting factor when using PLE for the extraction without changing the
isoavones prole of the sample. However, this may be considered
as positive in the case of hydrolytic methods (see Section 4). To date
no hydrolytic method using PLE has been reported and the potential changes in isoavone prole using high temperatures can be
explored in future investigations. Also, the inuence of the sample properties, such as particle size, protein content and enzymatic
activity and its relation with extraction efciency and isoavone
stability should be investigated in more detail.
5.1.2.3. Supercritical uid extraction. Supercritical uid extraction
is the process of separating one component (the extractant) from
another (the matrix) using supercritical uids as the extracting solvent. A supercritical uid is any substance at a temperature and
pressure above its thermodynamic critical point. They can penetrate samples of plant material almost as well as gases, due to
their high diffusion coefcients and low viscosity. At the same time,
their dissolving power is similar to liquids. Additionally, close to
the critical point, small changes in pressure or temperature result
in large changes in density, allowing many properties to be modied and to obtain selective extraction. The most commonly used
extracting agent is carbon dioxide (CO2 ), because of its low cost, low
toxicity, and easily reachable critical parameters (31.1 C/74.8 atm).
Furthermore, CO2 as a non-polar substance is able of dissolving
non-polar or moderately polar compounds. The addition of a polar
modier (e.g. MeOH) to supercritical CO2 (SC-CO2 ) is the simplest
and most effective way to modify the polarity of CO2 -based uids in order to increase the solubility of analytes. Modiers can
also overcome interactions between the analyte and the matrix,
increasing the extraction efciency of polar organic compounds
[113,114,132,141143].
Although SFE is one of the most complex technique for the
extraction of isoavones due to the high number of possible
variables and interactions, which can effect effectiveness, several
researchers successfully applied SFE to extract isoavones from different soy matrixes such as soy our, soy hypocotyls and soy cake, as
well as from other different matrices, like R. puerariae, M. recutita,
R. ofcinalis, F. vulgare and A. eupatoria L. [91,92,118,129,144,145].
An overview of the developed methods using supercritical uids
for the extraction of soy isoavones and evaluated parameters is
presented in Table 4.
As in most modern techniques and methods, stability of
isoavones under extraction conditions has not been studied so far.
This is important since relatively high temperatures are frequently
used. The same stability principles of the previously discussed techniques may apply to SFE and thus it is feasible to consider that
changes in isoavone proles can take place during extraction.
Therefore, evaluation of stability of isoavones using different SFE
conditions, such as temperature, duration and amount and type of
modier is urgently needed.
Regarding the methods developed so far, Chandra et al. [145]
tested a limited number of conditions with different pressures and
amount and type of modier for the extraction of some isoavones
(De and Ge) from various soy matrixes. The evaluation of the
extraction conditions revealed that at 50 C, 600 atm and 20% EtOH
extracted the highest amount of tested isoavones (nearly 93%).
It is worth noting that the development of the method was per-
formed spiking reference standards onto a lter paper strip which

was later extracted by SC-CO2 . The best-evaluated conditions were
used for the extraction of De and Ge from miso, tofu, and soy meal
and soy our using sample sizes ranging from 2 g to 10 g. Although
high recoveries were achieved, the method was limited in terms of
isoavones quantication.
Later, Rostagno et al. [118] evaluated the use of supercritical carbon dioxide for the extraction of soybeans isoavones (Gi,
Ge and De) using different temperatures, pressures and modier concentration. Maximum yield of Gi and Ge was obtained at
70 C/200 bar/10 mol%, while maximum yield of De was obtained
at 50 C/360 bar/10 mol%. For the extraction of Gi and Ge, a predominant effect of temperature was observed while for De, a
predominant effect of pressure was observed. Also a strong interaction between temperature and pressure was observed in the
extraction of the tested isoavones. The decrease in extraction efciency with the increase in the temperature can be explained by
the decrease in the supercritical uid density, while the decrease in
extraction efciency with the increase in pressure can be attributed
to a decreased uid diffusivity, which may affect interaction with
the sample. However, it is important to note that stability of
isoavones was not accessed, that only one glucoside (Gi) and
aglycones were measured and that malonyl glucosides were not
quantied. Since relatively high temperatures were used, it is plausible that degradation might have taken place during extraction
inuencing the obtained results. The authors suggested that enzymatic hydrolysis of Di might have occurred during extraction and
inuenced the results, since the best extraction temperature for
De was 50 C, close to the optimal temperature for the activity of
-glucosidases.
More recently, Kao et al. [91] modied the experimental conditions optimized by Rostagno et al. [118] and used 70% EtOH
as modier, instead of 70% MeOH and studied a similar range of
temperature and pressure for the SFE of isoavones (all 12 main
chemical forms present in soybeans) from soybean cake. The most
important aspect of this method, besides the high recovery (87.3%
when compared to stirring extraction), was the quantication of
all isoavone chemical forms, since it is the rst report of the
use of supercritical uids for the extraction of malonyl and acetyl
isoavones. The results showed that a maximum yield of malonyl
glucoside and glucoside was obtained at 60 C and 350 bar, while a
high level of acetyl glucoside and aglycone was produced at 80 C
and 350 bar. The highest yield of total isoavones was obtained
using 60 C/350 bar, possibly due to predominant concentration of
malonyl and glucosides in the sample. Although a different modier
was used, similar temperature and pressure interaction as reported
by Rostagno et al. [118] was observed.
However, as in most studies, stability was not evaluated and
results might be inuenced by degradation of malonyl and glucoside isoavones to their respective acetyl and aglycone forms. The
authors observed that, although using lower temperatures than
cooking and toasting, conversion or degradation can still occur
when in combination with pressure. The amount of malonyl glucosides declined after following a rise in the extraction temperature,
which was suggested to be related with solubility of these chemical
forms or to conversion to acetyl glucoside, glucoside or aglycone,
which may explain highest yields obtained at 80 C.
Arajo et al. [92] also tested different temperatures, pressures,
modiers, and modier concentration for the SFE of De and Ge
from soybean hypocotyls after hydrolysis. The highest yields of
these isoavones were obtained at 60 C, 380 bar using 3 cycles of
static and dynamic extraction of 15 min each with the addition of
10 mol% of 80% MeCN. Moreover, it was observed that modiers and
pressure variations have signicant effects in the extraction efciency. No isoavones were extracted without modiers and the
19
Table 4
Developed methods using supercritical uids for the extraction of soy isoavones and evaluated parameters.
of the method
Standards
Isoavones
Selected conditions
Reference
De and Ge
Extraction cell: n.e.

Temperature: 50 C
Flow rate: 9501000 mL/min
Restrictor temperature: 175 C
Rinse solvent: none
Extraction conditions:
400 atm and no modier
400 atm and 5% chloroform
400 atm and 5% MeOH
600 atm and 20% MeOH
600 atm and 20% EtOH
600 atm and 20% EtOH
[145]
Sample amount: 1 g
Extraction cell: 7.0 mL (reduced to
5.46 mL)
Inert material: glass stick
Modier: 70% MeOH
Gi, Ge and De
Dynamic cycle length: 20 min

CO2 ow rate: 1.0 mL/min
Extraction time: 90 min (3 30 min)
Trap: ODS
Rinse solvent: 1.5 mL MeOH
Rinse ow rate: 0.5 mL/min
Modier concentration: 0.5

and 10 mol%a
Temperature: 4070 C
Soybean hypocotyls
Di, Gi, Gly, De, Ge,

Gle, MDi, ADi, MGi,
AGi and MGly
De and Ge
Temperature: 5080 C

Fluxing solvent: 5 mL 50% EtOH
Pressure: 300400 bar

Rinse solvent: 1.5 mL 80% MeOH
Rinse ow rate: 0.5 mL/min
Soybean our
De and Ge

Inert material: glass beads

Soybean meal
Di, Gi, De and Ge

Extraction cell: 7.0 mL (reduced to
5.46 mL)
Inert material: glass stick
Modier: 70% MeOH
Separator 1: 8 0.3 MPa (40 C)

Separator 2: 6 0.3 MPa (30 C)
[118]
Sample amount: 1 g
Modier: 70% EtOH
Modier concentration: 10 mol%a
Soybean cake
50 C/360 bar, 10 mol%

(TIS and De)
70 C/200 bar, 10 mol%
(Gi and Ge)
Modier: MeOH, EtOH and

CH3 CN
Modier concentration: 0.5
and 10 mol%a
Temperature: 5070 C
Pressure: 1540 MPa

Temperature: 10100 C
Restrictor diameter: 25 and
50 m
Temperature: 4070 C
Pressure: 3060 MPa
Modier composition:
MeOH (60100%)
Modier concentration: 5.4,
7.8 and 10.2 mass%a
Malonyl glucosides,
glucosides and TIS:
60 C/350 bar
Acetyl glucosides and
aglucones:
80 C/350 bar
[92]
60 C/380 bar, 10 mol%

80% CH3 CN
[93]
35 MPa, 70 C, 5%
MeOH, 30 min, 50 m
[129]
40 C, 50 MPa,
9.80 kg/h, 80% MeOH at
7.8% mass, 2030
mesh, 200 min
[147]
CO2 ow rate:
3.929.80 kg/h
Sample particle size: 1060
mesh
AGi: acetyl genistin, AGly: acetyl glycitin, TIS: total isoavones, MeOH: methanol, EtOH: ethanol, CH3 CN: acetonitrile, n.e: not specied.
a
mol% of the CO2 mass passed through the system during the dynamic extraction.
20
predominant effect of the pressure in the amount of these two

isoavones (De and Ge) extracted was attributed to the likely
decrease of the steam pressure and increase in the density of uid
and a higher kinetics of desorption of the compounds from the sample matrix. As pressures increases, desorption is faster and more
solute is available for extraction. They also observed similar trend
as observed by Rostagno et al. [118], where an enhancement of
the extraction yield by the increase of pressure was dependent
of the temperature with correlation of pressure and temperature.
Also, major differences were observed for the assayed modiers.
Using 80% MeOH, 80% EtOH and 80% MeCN as modier, the relative amount of aglycones extracted were 9.61%, 11.27% and 25.65%
respectively when compared to stirring. It is clear that using the
proposed method 80% MeCN is much more effective than 80% EtOH
and 80% MeOH.
Another approach is to previously use SC-CO2 to enrich, or
clean, the sample matrix with isoavones by removing other
components from the matrix, as reported by Yu et al. [146], where
soy hypocotyls were defatted by SC-CO2 and used to produce
isoavone enriched soy protein.
Bajer et al. [129] optimized the extraction of De and Ge from
soy our using different pressures (1540 MPa), temperatures
(10100 C) and extraction times (1050 min). Optimal conditions
were 35 MPa, 70 C, modier MeOH (5%, v/v) and 30 min. The
authors reported clogging using restrictors of both 25 and 50 m
of internal diameter and placing the sample between glass beds
avoided the problem. Unfortunately, data obtained during the
method optimization was not provided, which could have been useful for future investigations on the use of supercritical uids for the
extraction of isoavones.
One of the latest applications of supercritical uid for the extraction of isoavones from soy matrixes was recently published by
Zuo et al. [147]. In this report the inuence of several extraction
parameters, such as pressure, temperature, ow rate, modier composition and concentration as well as sample particle size, was
evaluated for the extraction of some isoavones (De, Ge, Gi and
Di) from soybean meal. They observed that using specic conditions (40 C/50 MPa, 5.88 kg/h, modier ow rate of 0.6 L/h) higher
or lower amounts of water than 20% in MeOH (i.e. 80% MeOH)
extraction yield decreased, especially when using high MeOH concentrations (i.e. 90100%). This effect may be related with the
polarity of the supercritical CO2 which is excessively changed by
high or low amounts of modier. However, it does not exclude
the possibility that if extraction conditions were different (especially temperature and pressure), the highest yield modier could
have been achieved using a different modier composition since
extraction conditions may inuence CO2 polarity. The authors also
observed that using 80% MeOH, higher modier concentrations (i.e.
10.2 mass%) resulted in highest extraction yields, which can also be
explained by the changes in CO2 polarity. Here, extraction conditions can also inuence the effect of the modier concentration.
However, these results conrm the observations made by Rostagno
et al. [118], who also reported that higher modier concentration
(10 mol% of 70% MeOH) resulted in higher extraction efciency.
In most extraction techniques the increase of temperature
(which may be limited by stability of some isoavones) usually
increases extraction efciency due to several factors. This may not
be the case for SFE, since higher temperatures decreases CO2 density (maintaining pressure constant) and thus solvent power of the
uid. This issue has been demonstrated by Rostagno et al. [118] and
reects the results obtained by Zuo et al. [147], who observed that
increasing temperature from 40 C to 70 C decreased extraction
yield. Increased extraction temperature favors several processes
such as mass transfer, solubility and volatility of isoavones and
increase penetration power of SC-CO2 , but the positive inuence of
these factors may not be sufcient to counteract the reduced CO2

density. Also, the higher temperature may increase the solubility of
other sample components in the SC-CO2 and reduce the extraction
efciency of isoavones. Pressure (at 40 C) was also evaluated by
Zuo et al. [147] and a straightforward trend was observed, achieving highest yields using higher pressures, possibly due to increased
CO2 density. Increasing CO2 ow rate increased yields, obviously
due to increased mass of CO2 used. In this report, the most interesting extraction parameter evaluated (which has not been assessed so
far) was the inuence of sample particle size. Reducing particle size
from 1.19 mm (1020 mesh) to 0.68 mm (2030 mesh) improved
extraction yields and smaller particle size decreased extraction
yields. Smaller particles provide greater contact surface and better
allow the penetration of the SC-CO2 and consequently extraction
efciency increase. The explanation given by the authors to the
decrease of yields observed when using smaller particle sizes than
0.68 mm was the aggregate formation, which can cause the uid to
channel or short circuit.
One of the limiting aspects of this study was the use of one-attime strategy to optimize extraction conditions, which is not the
most recommended to be used for supercritical uids, since there
are much more variables and interactions that can more severely
inuence effectiveness than in other extraction techniques. The
best approach when using SC-CO2 seems to use experimental
designs or, preferably, a full screening of extraction conditions.
Also, the quantication of only glucosides and aglycones provide
only limited information on the effect of evaluated parameters
on the extraction of malonyl and acetyl glucosides, which have
been demonstrated to be the most difcult isoavone forms to be
extracted by supercritical uids.
There are a large number of variables that can affect the extraction using SFE, including not only pressure, temperature and type
and amount of modier, but also others such as the duration of
dynamic and static cycles, CO2 ow rate, thimble times swept,
restrictor temperature, trap composition, rinse solvent and rinse
ow rate. Trap composition and elution conditions (solvent, ow
rate and temperature) have not been studied so far and may
strongly inuence isoavone recovery and need to be investigated
in the future. Also the sample is an important factor since the particle size and interactions with the matrix (i.e. protein content)
may greatly inuence the ability of the supercritical uid to extract
target compounds. Other sample characteristics may inuence stability during extraction (i.e. glucosidase activity). The presence
of other sample components, such as oil, may interfere with the
amount of target compounds that can be extracted depending of
the uid density. Moreover, extraction conditions can have a great
impact on the effectiveness of a certain extraction parameter, and
selection of extraction variables should be carefully studied in order
to achieve maximum effectiveness. Therefore, much research is still
needed to fully determine the potential of supercritical uids for the
extraction of isoavones from soybeans and soy foods.
5.1.2.4. Microwave-assisted extraction. Microwaves are electromagnetic waves with wavelengths ranging from 1 mm to 1 m, or
frequencies between 300 MHz and 300 GHz. Microwave-assisted
extractions are based on absorption of microwave energy by
molecules of polar chemical compounds. The energy absorbed is
proportional to dielectric constant of the medium, resulting in rotation of dipoles in an electric eld (usually 2.45 GHz). The efciency
of MAE depends on several factors, such as solvent properties, sample material, the components being extracted, and, specically, on
the respective dielectric constants. The higher dielectric constant,
more energy is absorbed by the molecules and the faster the solvent
reaches the extraction temperature. In most cases, the extracting solvent has a high dielectric constant and strongly absorbs
microwave radiation. Microwaves may also promote selective and

rapid localized heating of moisture in the sample by microwaves.
Due to the localized heating, pressure builds up within the cells
of the sample, leading to a fast transfer of the compounds from
the cells into the extracting solvent. Additionally, by using closed
vessels the extraction can be performed at elevated temperatures
(above boiling point of the solvent) accelerating the mass transfer
of target compounds from the sample matrix [113,114,132,148].
This technique has been used only recently and on a few
occasions to extract isoavones from soybeans and from different matrixes, such as R. astragali, R. puerariae and peanuts
[86,110,124,149,150]. An overview of the developed methods using
microwaves for the extraction of soy isoavones and evaluated
parameters is presented in Table 5.
Since high temperatures are usually used by MAE is convenient to discuss the stability of isoavones during extraction before
detailing developed methods. There is apparently only one study
of soy isoavone stability during MAE, which was reported by Rostagno et al. [110]. They evaluated stability of soy isoavone extracts
(50%EtOH) at different temperatures (50150 C) for 30 min and
observed that temperatures above 50 C signicantly changed
isoavone prole of the extracts mainly due to conversions between
malonyl and their respective glucosides. Malonyl isoavones were
not detected above 100 C and temperatures higher than 125 C
promoted hydrolysis of glucosides to their respective aglycones.
Regarding the developed methods, several extraction solvents,
temperatures, and extraction solvent volume, as well as the sample size and extraction time were studied by Rostagno et al. [110]
for the optimization of an extraction protocol for all main soy
isoavones. In the rst selection of the most adequate extraction
solvent, pure solvents (MeOH, EtOH and water) extracted lower
amounts than 50% EtOH and 50% MeOH. Since 50% EtOH extracted
the highest amounts of total isoavones, it was further studied
in terms of water percentage (3070%) and results indicated that
using higher or lower amounts of water than 50% reduced extraction efciency. A similar result was obtained with solvent volume;
using high or low solvent volumes resulted in lower extraction efciency than intermediate volumes (2030 mL). After solvent and
volume were optimized, sample amount was evaluated revealing
that using low sample size reduce extraction efciency and sample size greater than 0.5 g does not improve yield. Therefore, the
authors concluded that a sample:solvent ratio of 0.5:25 (g/mL)
results in maximum efciency using the optimized conditions so
far. The effect of sample size is different when using MAE than with
other techniques, such as PLE, which increases extraction efciencies using smaller samples. Examining the effect of extraction time
the authors obtained a straightforward response: increasing extraction time increased extraction yield, and quantitative extractions
were obtained in 20 min. They also observed that most isoavones
present in the sample (approximately 75%) were extracted in the
rst 10 min of extraction. No isoavone degradation was observed
using the developed extraction protocol and a high reproducibility
was achieved (>95%).
The most interesting point of this report was the proposal of
an effectiveness factor in order to evaluate extraction conditions.
Most authors simply use total isoavones as reference of extraction
efciency. However, sometimes there is no signicant difference in
the total isoavone yield although there are signicant differences
between chemical forms, and selection of a certain solvent can be
complicated procedure. In the case of the proposed effectiveness
factor, it balances the extraction effectiveness for all isoavones
and more accurately identify the most adequate solvent.
Also recently, Careri et al. [86] adopted a fractional factorial design to develop a hydrolytic method for the extraction of
isoavonoid aglycones (Di, De, Gi, Ge, Biochanin A and Formonotin)
21
from yellow soybeans using microwave-assisted extraction. For

development of the method, several extraction parameters were
evaluated such as microwave power, extraction time, solvent,
extraction volume, acid concentration and re-hydratation time.
Several interactions among extraction variables were found and
the most relevant parameters resulted to be the microwave power,
the extraction time and the acid concentration. It is important to
note that sample was submitted to sonication for 15 min before
extraction using MAE, which may have extracted most isoavones
in the sample, as previously discussed, and the proposed method
can almost be considered a hydrolysis method rather an extraction
method.
It is clear that although highly efcient extractions can be
achieved with MAE, its potential is limited since only short extractions can be used due to isoavones stability. However, this may
be very useful when proposing hydrolytic methods, which can be
an interesting application for this extraction technique. Also, more
research is needed to determine the inuence of other parameters
not only on extraction but also on stability, such as pressure. Sample
characteristic such as humidity, -glucosidase activity, and particle
size also need to be investigated in future researches.
5.2. Liquid samples
Apart of solid samples, there are liquid soy samples that also
contain isoavones. Most common liquid soy foods are soy milk and
soy beverages blended with fruit juices. Usually, these samples are
freeze-dried and treated as solid samples, using the same methods
and techniques [41,44,4648,87,102,151,152].
The problem with the freeze-drying procedure is that it can take
days and may as well, increase variations on the determination of
isoavones, due to increased errors and degradation of the sample. Moreover, it goes in the opposite direction of the recent trend
of sample preparation that is to use fast methods and reduce to a
minimum the necessary steps from sample to analysis. It is not logical to have at hand an extraction method that can be accomplished
in 20 min and use a sample pretreatment of days.
Liquid samples are similar to a solid sample in that most
isoavones are in the suspended solids or already in the liquid
phase and therefore an extraction step can be used to extract the
isoavones from the solids and avoid freeze-drying the sample.
Indeed some authors successfully direct extraction of liquid samples without freeze-drying. Most authors used methanol (MeOH)
or ethanol (EtOH) with a sample:solvent ratio ranging from 4:1
to 1.6:1 (v/v) and extraction by reuxing or shaking for 14 h
[51,77,153,154].
These methods were adapted from protocols used for solid samples and more importantly, were not evaluated, very long extraction
times were used or only a few isoavones were studied. Also, the
use of reuxing causes malonyl isoavones to undergo degradation
to the respective glucosides and aglycones, changing the isoavone
prole of the samples and limiting the information obtained. On the
other hand, they indicate that an extraction step can used instead of
freeze-drying the sample. This was demonstrated by Rostagno et al.
[151], who recently developed a new method for the fast determination of isoavones from soy beverages blended with fruit juices
using UAE.
During the method development, several parameters were
studied such as solvent (methanol and ethanol), sample:solvent
ratio (5:1 to 0.2:1), temperature (1060 C) and extraction time
(530 min). The most important parameter for the extraction of
isoavones from soy drinks was the sample:solvent ratio. Also, samples were freeze-dried, extracted using a reference method and
compared with the optimized method and no signicant difference was observed on total and individual isoavone concentration.
22
Table 5
Developed methods using microwaves for the extraction of soy isoavones and evaluated parameters.
of the method
Isoavones
Di, Gi, Gly, De, Ge, Gle,

MDi, MGi, MGly, ADi,
AGi and AGly
Fixed extraction
conditions
Solvent: 25 mL
Microwave power:
500 W
Magnetic stirring: 50%
nominal power
Solvent:
Water
EtOH, 50% EtOH
MeOH, 50% MeOH
EtOH (3070%)
Selected conditions
Reference
0.5 g, 50 C, 20 min and

50% EtOH
[110]

Solvent volume: 1535 mL
Soybeans
Di, De, Gi, Ge, Biochanin

A and Formononetin

Sonication before
extraction: 15 min
Microwave power: 10 and 600 W

Extraction time: 1 and 8 min
Solvent: 80% CH3 CN and 80% MeOH
Solvent volume: 3 and 8 mL
600 W, 1 min, 3 mL of
80% CH3 CN, 12 M HCl
and no re-hydratation
time
[87]
Acid concentration: 1 and 12 M

Re-hydratation time: 0 and 10 h
AGi: acetyl genistin, AGly: acetyl glycitin, MeOH: methanol, EtOH: ethanol, CH3 CN: acetonitrile.
The novelty of this work resides in its simplicity and rapidity when
treating a troublesome liquid sample without the need of freezedrying the sample before extraction. This report provides valuable
information although further evaluation of the inuence of other
extraction parameters, such as sample characteristics, ultrasound
frequency and power and the use of ultrasonic pulses is still needed
and will likely be explored in future investigations.
Another sample preparation technique that can be used for
extracting soy isoavones from liquid foods is solid phase extraction. SPE involves adsorption of sample components on the surface
of a solid sorbent, followed by elution with a selected solvent. A
variety of sorbents available in the market allows not only the isolation of analytes, but also the removal of interferences. However,
the whole potential of this technique for the analysis of isoavones
in foods is yet to be determined. Although SPE applications for the
analysis of isoavones from blood, plasma, urine and serum are
relatively common [155161], only a few works explored the SPE
potential for the analysis of isoavones from liquid samples.
Mitani et al. [162] for instance, proposed an automated on-line
in-tube solid phase microextraction (SPME) coupled to HPLC for
the determination of daidzein and genistein in soy foods. In-tube
SPME is a preconcentration technique using an open tubular fusedsilica capillary with an inner surface coating as the SPME device,
which can be easily coupled on-line with HPLC. In tube SPME allows
for convenient automation of the extraction process, which not
only shortens the analysis time, but also provides better accuracy,
precision and sensitivity relative to off-line manual techniques.
However, a hydrolysis step was required because the isoavone glucosides present in the sample were difcult to concentrate using
such conditions, which limit its usefulness for quantication of all
isoavone chemical forms. Moreover, since most isoavones are
in the suspended solids in liquid samples, it is very likely that an
extraction step before the in-tube SPME method will be required;
otherwise it will only separate isoavones present in the liquid
phase and may lead to an underestimation of isoavone concentrations.
Therefore, the greatest potential of this technique is the concentration and clean-up of extracts coupled to an extraction technique
such as PLE or SFE, or after the extraction procedure with one of the
previously discussed methods. It also may be used before extraction
to eliminate undesirable components of the sample and allowing a
more selective extraction of target compounds.
5.3. Optimization of extraction conditions

In view of the fact that chemical modications of isoavones
may occur during the extraction process, not only isoavone extraction efciency for a particular solvent need to be considered when
comparing the extraction solvents, but also preservation of original isoavone composition during extraction, minimizing chemical
transformations.
Concentrations of -glucosides and acetyl glucosides forms
could be increased or decreased during extraction procedure, and
aglycones could be increased as a consequence of chemical transformations. Higher amounts obtained of these chemical forms do
not necessarily mean higher extraction efciency since it could be
result of the transformations. Thus, it is difcult to determine the
most efcient solvent for extracting -glucosides, acetyl glucosides
or aglycone isoavones by simple comparison of yields. Malonyl
isoavones are the chemical forms most susceptible to degradation
and therefore the higher amount of malonyl glucosides present in
the extracted material indicates either higher extraction efciency
of the solvent, better protection from chemical transformations or
both. In the case of not quantifying malonyl isoavones the best
choice is to determine the stability during extraction.
Apart of stability issues, extraction conditions should be
optimized for each solvent and for each sample. Quantitative
recoveries can be achieved with most commonly used solvents for
the extraction of isoavones from soybeans and soy foods, given
extraction conditions are optimized enough. Therefore, the discussion of which is the best solvent should be addressed in terms
of advantages and disadvantages. For example: MeCN sometimes
can extract more isoavones than MeOH or EtOH. However, MeCN
have a higher cost, toxicity and lower environmental compatibility
than EtOH and MeOH and if quantitative recoveries are achieved
with these solvents there is no justication for the use of MeCN
as extractant. Also, the inuence of the sample should not be
overlooked. It can seriously affect the ability of a given solvent
to extract some isoavones and use a method optimized with a
certain sample should not be used without evaluation even for a
similar sample. An internal standard can be used to account for
losses during extraction for quantication studies. An ideal internal
standard should be a compound structurally related to the analyte
and with a similar polarity, but with a retention time that does
not overlap the other peaks during the chromatographic analysis.
Spiking the sample may also be used to estimate if quantitative

extractions are achieved, but enough time should be given for the
standards interact with the sample. However, interaction with the
sample matrix cannot be ensured even if prolonged soaking is
used. Also, it may be of interest to use an extract obtained with
optimized conditions rather than pure standards, since an extract
more closely resemble the sample. Finally, sequential extractions
are seriously recommended to ensure that quantitative recoveries
are achieved [106,107,109,151].
Overall, to optimize extraction conditions three strategies can
be used: one-variable-ata-time optimization, the use of an experimental design, or a combination of both. The one at a time
approach is more objective and can be used to isolate the effect
of a given variable and provide easier interpretation. The drawback of this strategy is that it is somewhat limited to observe
interactions among extraction variables. For instance, when optimizing an extraction using PLE, MeOH extracted higher amounts
of isoavones than EtOH at 60 C and 100 bar of pressure. However,
if higher temperature or pressure was used, the physicalchemical
properties of the solvents change and MeOH may not obtain higher
yields than EtOH. To observe interactions, either a screening of all
possible extraction conditions or an experimental design can be
used. Screening implies a huge number of extractions, which takes
time and have high cost. The experimental design allows reducing the number of analysis needed to identify the most important
extraction variables. An excellent optimization strategy would be
to use an experimental design to identify the most important variables, and further investigate these variables in detail by screening.
Also, the use of mathematical models and computer aided optimization may be valuable tools for reducing the time required to
develop extraction methods hampered by the existence of a great
number of variables inuencing the process and will likely explored
in the future.
5.4. Critical comparison of extraction methods
Serious efforts have been made in the last decade trying to
compare techniques, methods and solvents for the extraction of
soy isoavones. A relative comparison of extraction techniques/
methods available in the literature is shown in Table 6.
Ascertaining the most suitable extraction technique/method/
solvent for determination of isoavones in soy samples is relatively difcult. In spite of the fact that the substances investigated
are quite close chemically, physically and physiologically, there are
important differences on the extractability of each chemical form
and isoavone type, and therefore it is almost impossible to suggest a single extraction solvent that ensures that all isoavonoids
are extracted with maximum yields from all types of soy samples
[107,129]. It is also important to remark that, in most cases, what
are being compared are extraction methods using different extraction techniques and not the extraction technique itself. Stability
may also affect results and provide incorrect relative comparison
values.
Rostagno et al. [118], for example, developed an extraction
method for the extraction of some isoavones from soybean
our using SFE and compared the results obtained with different techniques (UAE and soxhlet). Highest yield of total isoavones
evaluated were obtained by UAE, followed by soxhlet and SFE methods. Soxhlet extracted 70%, and SFE extracted approximately 30%
of the amount obtained by the UAE method, respectively. However, there are several factors that may responsible for the observed
differences among extraction methods that may be attributed to
the extraction method rather than to the technique itself. In this
case, the most important is that stability was not evaluated and
only a few isoavone chemical forms were quantied. This is of
23
uttermost importance since using soxhlet extraction, degradation

of isoavone malonyl, acetyl and glucoside forms is known to take
place increasing concentration of glucosides and aglycones and
therefore results may not be comparable. Also, performing UAE
for 90 min is very likely to increase extraction temperature and
on similar basis and promote degradation of isoavone conjugate
forms. Therefore, these results should be taken with caution and
the relative efciency of SFE may be underestimated.
The comparison of extraction methods can be very relative,
especially regarding the method used as reference. Arajo et al.
[92] compared SFE and stirring with for the extraction of some
isoavones from soybean hypocotyls. The proposed SFE method
extracted 25.65% of the aglycones extracted by stirring. The relative
amount extracted reported is overestimated since the hydrolysis
was only applied to samples extracted by the SFE technique and not
to the reference method (stirring). Hydrolysis markedly increases
the concentration of aglycones present in the sample at the expense
of the glucosides and hence increases the available amount of aglycones to be extracted and the actual yield may be much lower. Also,
the necessary corrections when using hydrolytic methods were not
applied which further inuence results obtained.
The authors go even further and claim the results obtained with
the proposed method were superior to those obtained by Chandra et al. [145] and Rostagno et al. [118]. The yield of aglycones
obtained by Arajo et al. [92] was 180 g/g while Chandra et al.
[145] extracted between 15 g/g and 103 g/g dry weight (depending of the sample) and Rostagno et al. [118] extracted 32.6 g/g.
A key point for the differences between these reports is also the
hydrolysis of the extracts used by Arajo et al. [92]. Comparing
yields of only aglycones between hydrolytic and non-hydrolytic
methods is rather complicated and even more is the non-hydrolytic
methods used as reference quantify only a few glucosides [118] or
no glucosides at all [145]. If all chemical forms are quantied it
is possible to compare hydrolytic and non-hydrolytic methods by
making the corrections for the molecular mass. Moreover, different
samples and sample types were used. Araujo et al. [92], extracted
isoavones from soy hypocotyls, Chandra et al. [145] from miso,
tofu, soy meal and soy our and Rostagno et al. [118], from soy
our. The concentration of a given compound on the sample directly
affects the yields and comparison of different samples should be
based on relative recoveries rather than in yield. Also, sample stability may be affecting the recoveries of the earlier reports, thus these
results may not be comparable in the same basis. Therefore, authors
should be very careful when making assumptions when comparing results with those obtained with other methods available in the
literature.
Kao et al. [91], compared SFE and shaking for the extraction of
isoavones from soybean cake and observed that shaking extracted
higher amounts of total isoavones (approximately 35%) than the
highest amounts obtained with SFE (60 C/350 bar). However, SFE
extracted higher amounts of acetyl glucosides and aglycones. At
60 C/350 bar SFE extracted approximately 33% and 91% of malonyl and glucosides, respectively than the amount obtained with
shaking, while shaking extracted 80% and 87% of the acetyl and
aglycones, extracted by SFE. At 80 C/350 bar, SFE extracted even
lesser malonyl and glucosides (17% and 70%, respectively) than
shaking, and shaking extracted even less acetyl and aglycones
(65% and 58%, respectively) than SFE. These observations may have
been inuenced by degradation of malonyl isoavones and without
proper evaluation of isoavone stability results might give a false
impression of effectiveness.
Also, the relative yield obtained with the developed method
(65%) is much higher than those obtained by Rostagno et al. [118]
(28%) and Arajo et al. [92] (26%). However, the relative yields
reported by Rostagno et al. [118] (28%) and Arajo et al. [92] were
24
Table 6
Relative comparison of extraction techniques/methods.
Sample
Isoavones
Compared techniques/methods
Relative yield (%)a
Reference
Soy our
Soybean cake
Gi, Ge and De
Di, Gi, Gly, De, Ge, Gle, MDi, MGi,
MGly, ADi, AGi and AGly
De and Ge
Di, Gi, De and Ge
Di, Gi, Gly and MGi
Di, Gi, De and Ge
Di, Gi, Gly, Ononin, De, Gle and Ge
Gi, MGi, AGi and Ge
SFE/UAE/Soxhlet
SFE/Shaking
28/100/68
74/100
[118]
[92]
SFE/Stirring
SFE/Stirring
UAE/Stirring
PLE/UAE/Soxhlet/Shaker/Vortex/Stirring
26/100
87/100
100/85100b
100/93/68/71/66/70
[93]
[147]
[106]
[134]
PLE/UAE/Soxhlet/PLE + UAE
UAE/Soxhlet/PLE + UAE
PLE/Stirring
49/14/64/100
22/68/100
98100/88100c
[135]
[137]
[133]
UAE/UHOM/SFE/PLE/Soxhlet
MAE/UAE
100/93/16/71/69
100/100
[129]
[110]
Soybean hypocotyls
Soybean meal
Soybeans
Soybeans
Soy bits
Soy bits
Soy our, Meat substitute,
nuts and protein isolate
Soy our
Soy our
De and Ge
AGi: acetyl genistin, AGly: acetyl glycitin, UHOM, Ultrasonic homogenizer.
a
Relative to the technique which extracted the highest amount of total isoavones.
b
Depending of the solvent used.
c
Depending of the sample used.
inuenced by degradation during extraction with the reference

method and by the use of hydrolytic methods. Since stability was
not accessed by Kao et al. [91] the relative yield, although much
higher than those reported earlier, is only speculative.
Zuo et al. [147], achieved even higher relative recoveries (87%)
than Kao et al. [91] when compared to solvent extraction using
magnetic stirring using different extraction conditions. In this case,
only a few isoavones were quantied (glucosides and aglycones)
and results were reported in only in terms of total isoavones and
the issues of the previous discussed method apply.
Stability during extraction may be one of the most important
aspects when comparing extraction techniques/methods since they
may change isoavone prole of the sample and affect yields, which
is especially important when all isoavone chemical forms are not
quantied. For the reliable comparison of extraction techniques,
extraction conditions should be optimized for each technique,
ensure that using the optimized conditions do not affect isoavone
prole and only then, results might be comparable. One option to
obtain comparable results between methods/techniques is the use
of hydrolytic methods, including the reference method, since it will
eliminate variations derived from transformations. The handicap
of using hydrolysis is that only limited information is obtained (i.e.
total isoavones) although it may prove useful in some cases.
Several other authors tried to compare different extraction techniques/methods. Rostagno et al. [106] compared UAE and magnetic
stirring using several different solvents (water, EtOH, MeOH and
MeCN with different water percentages) at 10 C for 10 min and
observed that UAE extracted between 0% and 15% more isoavones
than magnetic stirring at 10 C, depending of the solvent. At 60 C
similar increase in extraction efciency was observed. Also, a similar solvent response was observed using magnetic stirring and
UAE, achieving maximum extraction efciency using solvents with
4060% water.
Luthria et al. [134] compared several extraction techniques (stirring, shaker, UAE, vortexing, soxhlet and PLE) of 12 main isoavones
from soybeans using the same solvent (DMSO:EtOH:water
(5:70:25) as extracting solvent. PLE was the most effective method
for the extraction of total isoavones, extracting between 30% and
35% more isoavones than the other methods. Total isoavones
extracted by UAE was 93.3% as compared to PLE and shaking
extracted 75.6% of amount extracted by UAE. Both, the total
isoavone content and the isoavone HPLC prole varied signicantly with different techniques. MGly and De were detected only
on PLE and UAE extracts. Shaking and stirring extracted the highest
amounts of malonyl isoavones (MDi and MGi) while PLE extracted
the highest amount of acetyl glucosides. Extraction conditions
(sample size, extraction length, number of extraction cycles and
temperature) used in the PLE procedure were point by point optimized by Rostagno et al. [109] while the other extraction methods
were not, and therefore is not surprising that PLE revealed to be the
most effective extraction technique.
Klejdus et al. [135] evaluated different techniques/methods (PLE,
UAE, soxhlet and PLE + UAE) for the extraction of isoavones (De,
Ge, Di and Gi) from soybean foods. PLE + UAE (1 min sonication)
extracted the highest amount of isoavones followed by soxhlet,
PLE and UAE, in this order. Soxhlet extracted the highest amount
of aglycones while PLE + UAE extracted the highest amount of glucosides. However, malonyl isoavones were not quantied and
stability was not accessed and the highest amount of isoavones
extracted by PLE, PLE + UAE and soxhlet than by UAE alone may be
partially attributed to degradation of malonyl isoavones leading
to their respective glucoside and aglycone forms.
Later, Klejdus et al. [137] evaluated soxhlet, UAE and PLE + UAE
for the extraction of isoavones from soy bits and obtained similar
results. In this case, however, sonication time before PLE was 5 min
instead of 1 min. The same stability issues of the later report also
apply here.
Downing et al. [133], compared PLE and the method developed
by Barnes et al. [78] using stirring. The method by Barnes et al.
[78] required 60 min, while the PLE procedure (performed at 80 C)
required 20 min to extract similar levels of genistein equivalents.
They observed signicant differences on the extraction of conjugated forms of genistein extracted by these two methods. Heat
during PLE caused signicantly less acetyl genistin to be present
in the extracts when compared with stirring where ambient temperature was used. However, this outcome was dependent of the
sample. In some soy our samples deesterication occurred and in
others not. Acetyl genistin was much more susceptible to degradation than malonyl genistin and degradation of the former only
occurred in one sample (soy nuts). The change in the forms of
genistein was attributed to heat-induced deesterication of the
acetylgenistin and malonyl genistin to genistin.
Bajer et al. [129] also evaluated different extraction methods
(UAE, ultrasonic homogenizer, SFE, PLE and soxhlet) for the extraction of De and Ge from soy our using optimized conditions. They
observed that the different isoavones present in the assayed
samples are extracted in maximum yields by different methods.

The highest amount of these two isoavones was obtained by UAE,
followed by ultrasonic homogenizer, PLE, soxhlet and SFE, in this
order. No signicant difference was observed between PLE and
soxhlet. For De, the best extraction methods were UAE and soxhlet
followed by ultrasonic homogenizer, PLE and SFE, in this order,
while for Ge the extraction methods with highest yields were
ultrasonic homogenizer, UAE, PLE, soxhlet and SFE, in this order.
However, these results should be taken with care. Stability was
not assessed nor was the malonyl and glucoside forms quantied.
Soxhlet extraction is known to promote degradation of these forms
and nevertheless, extracted low amounts of Ge (more than half of
the amount extracted by ultrasonic homogenizer). Moreover, no
data on the optimization of each extraction method was provided
and therefore these may be rather speculative.
There is a fundamental difference when comparing the extraction of a given compound, comparing the extraction technique
(UAE, PLE, etc.) and the extraction method. Using the same technique is possible to have two different quantitative extraction
methods. In most cases, authors compare different extraction techniques using different extraction methods and therefore to draw
conclusions from this kind of reports is difcult. If the aim of the
study is to optimize an extraction method using a certain technique
and make a comparison with other methods (that use different
extraction techniques), it is essential that authors, use optimized
conditions for all extraction methods and ensure that they do not
affect isoavone prole of the sample. Comparison with a reference
method reported in the literature may also be used.
An illustrative example for the comparison of the different
extraction methods/techniques can be taken from the work of Rostagno et al. [110]. These authors proposed an MAE method after
optimizing several extraction conditions and evaluated isoavone
stability with the optimized method, which did not affect isoavone
prole in the sample. The MAE method was compared with a previous developed method using UAE and no difference was observed in
total and individual isoavone yields. With both methods, quantitative extractions were obtained in 20 min. In this case, not only are
the methods comparable but also the extraction technique, which
are similarly effective for the extraction of isoavones from soybeans. Moreover, quantitative recoveries are achieved with both
techniques without changing the isoavone prole of the sample.
Altogether, SFE seems to be less efcient for extraction of
isoavones than other techniques, while UAE, PLE and MAE, the
most efcient. Apart from extraction efciency, there are other
aspects that are important when determining the most suitable
extraction technique. Selection of an appropriate extraction technique entails consideration of not only the recovery but also the
cost, time of extraction, and the volume of solvent used, among
others. From the point of view of solvent consumption, SFE is without doubt the best extraction technique. In contrast, soxhlet require
large amounts of solvent and is a time-consuming procedure and
may affect isoavone prole. UAE and MAE have demonstrated to
be fast techniques for the extraction of isoavones from soybeans,
followed closely by PLE. SFE is an intermediate option. Also, the
need of post-extraction purication steps is an important issue. PLE
and SFE provide sufciently pure extracts without the need of subsequent ltration, while using UAE the ltration step is required.
PLE has been shown to have important advantages over competing techniques as regards time saving, solvent use, automation and
efciency. PLE and SFE have the advantage that no ltration step is
needed, since the matrix components that are not dissolved in the
extraction solvent may be retained inside the sample cell. Also, it
allows to easily performing reextractions of the sample to ensure
quantitative extractions are achieved. PLE and SFE are very convenient for the purposes of automation and on-line coupling of the
25
extraction and separation techniques. Also, PLE and SFE offers the
possibility of performing the extractions under an inert atmosphere
and protected from light, which represents an attractive advantage
since many compounds, are sensitive to these two external factors
[67,109]. However, some modern extraction techniques (MAE, PLE
and SFE) are not always available in the average laboratory, due to
the high cost of the equipment.
For the analysis of a particular sample with approximate knowledge of concentration and distribution of isoavones, such as for
routine quality control of similar soy ours, UAE can be used due
to its low cost and high efciency. In contrast, for the analysis
of different samples with unknown isoavone concentration and
distribution, PLE may be preferred since besides high efciency
it allows to easily perform reextractions of the sample. Thus, the
choice of an extraction technique will depend of several factors
besides efciency. Among these factors, implicit characteristic of
the techniques are particularly relevant such as instrumental cost,
level of automation and possibility of on-line coupling with analysis
technique. A good way to select an appropriate extraction technique
is to consider practical aspects and establish a multicriteria decision
making procedure using desirability function optimization.
6. Post-treatment of extracts
After extraction of isoavones from the sample matrix is performed, the extract can be submitted to a series of post-treatment
steps before the analysis. These procedures can be reduced to a
minimal depending of extraction technique used. After extraction,
insoluble materials are usually removed by ltration or centrifugation and sometimes, the extract are immediately analyzed without
further preparation. If extract is obtained using PLE or SFE ltration and centrifugation is not required. Also, several authors simply
pass the sample through 0.45 m lters after extraction and avoid
the centrifugation step [86,91,109,110,134,144,147,151]. The limitation of not using centrifugation is the difculty of correcting the
sample volume and solvent losses during ltration, especially with
small samples. This problem can be prevented by using an internal standard with the specic aim of correcting the sample volume
[81,106,109,110,151].
After ltration, liquidliquid extraction can be used to remove
undesired sample components such as the lipophilic components,
in order to preserve reverse phase chromatographic columns.
Hydrolysis of the extracts can also be used after the extraction using
the same methods discussed in Section 4.
Another common post-extraction procedure is the partial or
complete removal of the solvent by rotary evaporation and redissolution of the sample either on the mobile phase used for the
chromatographic analysis or in 80% MeOH [41,42,72,90,102,107,129,
133,135137,144,152].
This procedure can be used to pre-concentrate the extracts and
reduce detection and quantication levels during chromatographic
analysis. Another reason for this post-extraction step is to avoid
the peak distortion caused by injecting samples containing high
concentration of MeCN onto columns equilibrated with low MeCN
concentration. The procedure is time-consuming and such handling always increase variability and can be source of losses and
degradation. Moreover, the use of this post-extraction step can be
avoided by using a compatible solvent for extraction such as EtOH
or MeOH, by limiting the sample size to less than 5 L when using
conventional C18 columns or by using high ow HPLC methods with
monolithic columns [81,104]. Avoiding the use of such cumbersome procedure can greatly decrease the time required for sample
preparation.
More often, some additional sample preparation are used to isolate analytes of interest from other sample components that can
26
interfere with the chromatographic analysis or as an extract enrichment step, wherein the analyte concentration is increase above
the determination limit of the nal determination technique. SPE
is one of the most used enrichment techniques. SFE have been
used by some authors [52,129,163,164] to provide a clean concentrated isoavone extract to be used in the chromatographic
analysis.
A wide selection of sorbents, ranging from classical C8 and C18
silica based sorbents to new polymeric materials, enables substantial selectivity of the enrichment process. Most traditional solid
phase extraction sorbents often result in poor analyte recoveries,
insufcient cleanup, or irreproducibility from extraction to extraction. Polymeric sorbents are the latest breakthrough in SPE, since
they enable higher recoveries, higher reproducibility, and lower
consumption of plant materials for the HPLC analysis than classical
sorbents. Polymeric sorbents also have the advantage of remaining conditioned even if the sorbents accidentally run dry during
the extraction. Divinylbenzene based polymeric sorbents exhibit
excellent stability over the whole pH range unlike classical modied silica gel sorbents C18 and C8 [164]. Excellent results were
obtained using polymeric sorbents for concentration and clean-up
of isoavones from red clover [164,165] and soy extracts [126]. For
soy isoavones, divinylbenzene based polymeric cartridges showed
better retention and much higher breakthrough volume during
sample load and washing steps than classical C18 sorbents from
different manufactures.
Besides of the use of new polymeric sorbents, the recent trend
for the use of SPE is automation and coupling on-line with the analysis method. Compared with manual methods, automated SPE is
less labour intensive, requires less sample handling providing better recovery, is more reproducible, is performed in a closed system
(less chance of sample oxidation or solvent evaporation) and can be
performed relatively fast. For instance, Rostagno et al. [126] developed an automated SPE method for soy isoavones achieving very
high recoveries (99.37%) and reproducibility (>98%) with a concentration factor of approximately 6:1 in less than 10 min. Another
future prospect for the use of SPE is to be coupled on-line with the
extraction (such as PLE and SFE) and analysis methods reducing
to a minimum post-treatment of extracts in order reduce sample
handling allowing more precise and reliable data to be obtained.
7. Separation approaches/techniques
Many different analytical methods can be used for the analysis
of isoavones from soybeans and soy foods. These analytical methods include gas chromatography, liquid chromatography (both with
and without mass detectors) capillary electromigration techniques
(CE), and immunoassay. In the last few years several reviews about
the analysis of isoavone extracts using these techniques have been
published [166171].
Chromatography and CE are, without doubt, the most relevant
techniques applied in this eld. The use of CE for the analysis of
soy isoavone samples is very attractive due to the high resolution,
efciency and analysis speed with minimum reagent and sample
consumption. There are a variety of versatile CE separation principles which are feasible of adapting to solve different analytical
problems. The possibility of coupling CE to different types of detectors, especially to sensitive electrochemical detectors, is one of the
main advantages of these techniques and point to a powerful tool
for the characterization of isoavones in soy derived samples.
Although the use of CE in the identication process is a potentially appropriate means of rapid screening it has been applied only
on a few occasions for the analysis of isoavones from soy samples, and in most cases only some chemicals forms were identied
[168,171174].
However, CE is characterized by poor quantitative reproducibility, mainly caused by inconsistent ow rate and injection volume
or amount. Although signicant advances in this aspect has been
made, the reproducibility issues of CE, especially when applied to
real samples, still needs to be solved before it become a real alternative to more consolidated techniques such as chromatography
[171,175].
In this context, of all available analysis techniques, HPLC is
the method of choice since it requires simple pre-analysis sample
preparation, allows measurement of all isoavone chemical forms,
is highly efcient and reproducible, is widely available and has been
extensively studied. HPLC separation of isoavones is generally carried out on reversed-phase columns with using MeOH or MeCN
and water containing a small amount of acid (formic, acetic, phosphoric or triuoroacidic acids) as mobile phase. Since isoavones
exhibit a weak acidic nature the use of acids can make the analytes
to be easily dissociated in a solvent system enhancing chromatographic separation, resolution and improve peak shape [169]. Most
often used detectors coupled to HPLC are UV and UV-diode array
detection (DAD) monitoring in the range of 230280 nm, since all
isoavones exhibit an intense absorption in this UV region of the
spectrum. Gradient elution is usually necessary in order to separate all main isoavones since they are very chemically close. As
previously mentioned some isoavones are particularly difcult
to separate from each other (i.e. MGi, AGly and De) [81] and isocratic elution has proven to be insufcient. Isocratic elution may be
accomplished if a hydrolysis step is used before analysis with the
implicit handicap of quantifying only the aglycone forms [51].
Conventional microparticulate 5 m RP-C18 columns are the
most used stationary phase and analysis time needed to separate
all main soy isoavones usually reach 60 min. Similarly to sample preparation, the current trend for the analysis of soy isoavone
extracts is toward fast, high sensitive and high-resolution separation of all main chemical forms of these compounds in soybeans
and soy foods.
One alternative to achieve faster and more sensitive analysis is
to reduce the particle size of the stationary phase. The use of smallparticle columns (less than 2 m particle size) can shorten analysis
times, while maintaining or even increasing high separation efciencies, since it is very well known from Van Deemter equations
that the efciency of chromatographic processes is proportional to
particle size decrease and to the higher allowed linear velocities.
The negative aspect of small particle packed columns used in HPLC
is the higher column back-pressure generated [176,177]. Hence, to
take full advantage of sub-2 m particles stationary phases high
pressure liquid chromatography systems that operate at high pressures (>400 bar) are required. Not only is the system capacity of
operating at high pressures important but also the ability to accurately and reproducibility integrate an analyte peak and detector
sampling rate must be high enough to capture enough data points
across the peak. Some applications of this innovative technology for
isoavones from different matrixes can be found in the literature
[178181].
Churchwell et al. [178] compared UPLCMS conventional
HPLCMS for the determination of isoavones in waste water and
found that in general, UPLCMS produced signicant improvements in method sensitivity, speed, and resolution when compared
to conventional HPLCMS. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak and
from a separation of diastereomers not possible using HPLC.
As an example of the enormous potential of these new advances
in chromatography, Klejdus et al. [179], developed an analysis
method for some selected isoavones (Di, Gly, Gi, De, Gle, Ge,
Ononin, sissotrin, formononetin and biochanin) which takes less
than 1 min. The method was successfully applied to soy bits and red
clover extracts with excellent results. Moreover, Klejdus et al. [180],

accomplished simultaneous separation of not only isoavones but
also together with several phenolic acids in less than 2 min. Further investigations however, are still needed to evaluate the use
of small particle columns for the separation of malonyl and acetyl
isoavones that are the most troublesome compounds to separate
in soy extracts.
Another alternative to perform high-speed separations using
liquid chromatography is the use of monolithic columns. As compared to particle bed columns, monolithic columns represent a
single piece made of porous cross-linked polymer or porous silica.
Monoliths are made in different formats as porous rods, generated
in thin capillaries or made as thin membranes or disks. The major
goals of applying monolithic columns in HPLC were to achieve low
column backpressure and fast mass transfer kinetics [182,183].
Major chromatographic features of monolithic silica columns
arise from the large through-pore size/skeleton size ratios and high
porosities, resulting in high permeability and large number of theoretical plates per unit pressure drop. High permeability and small
diffusion path length provided by the presence of large throughpores and relatively small-sized skeletons resulted in the lower
plate height and the lower pressure drop with monolithic silica
columns compared with a particle-packed column. With lower column backpressure it is possible to increase solvent ow rate making
faster separations possible with current instrumentation [182].
Monolithic columns have been used in some occasions for
the analysis of isoavones in soy extracts [81,110,126,151,184,185].
Apers et al. [184], achieved separation of most soy isoavones
(except malonyl glucosides) from soy extracts in less than 18 min.
Further, Kim et al. [185] recently proposed another method using
monolithic columns for the analysis of four isoavones (Gi, Ge, Di
and De) from soybeans and soybean pastes in 7 min.
To date, the fastest separation of all main isoavones in soy
extracts was obtained by Rostagno et al. [81]. After optimization of
analysis conditions using monolithic columns all isoavones were
resolved in less than 10 min using acidied MeOHwater at a ow
rate of 4 mL/min. Such high solvent ow rate illustrates the low
pressure obtained with this type of column.
However, the use of MeOH in the mobile phase to perform fast
separation has some limitations when compared to MeCN, since it
has a higher viscosity resulting in higher pressure, which can reduce
maximum solvent ow rate allowed within the chromatographic
system maximum pressure and increase separation time required
for all isoavone chemical forms. Therefore, it is feasible to assume
than even shorter analysis run times than 10 min can be achieved
either with monolithic columns with MeCN or with small particle
columns using UPLC systems and that more research is needed in
this direction.
To perform fast HPLC analysis, aside the chromatographic system, the stationary or mobile phase, the simplest approach consists
in operating columns at higher temperatures. Mobile phase viscosities decrease rapidly with increasing temperature; the column
efciency is barely changed but the optimum velocity increases
markedly, allowing the same resolution to be obtained much faster
but with nearly the same inlet pressure [186]. In this case, some of
the new small particle columns have a clear advantage over conventional 5 m C18 and monolithic columns since they can operate
at much higher temperatures, reaching 90 C depending of pH conditions.
8. Conclusions
In the last two decades considerable efforts were directed to
quantify isoavones in soybeans and derived foods generating several sample preparation and analysis methods which resulted in
27
huge amounts of scattered information. Although a critical view of

these methods is given in this review, is important to point out that
they contributed to better understanding of the complex task that
is isoavone determination and that the necessary steps are being
given in the direction of achieving reliable and precise information
about the distribution of these compounds in foods.
Several aspects of sample conservation and sample preparation for the determination of soy isoavones should be carefully
observed since they are an important source of misinformation. Sample conservation is particularly important since some
isoavones are relatively unstable and adequate storage conditions
are necessary to preserve the original prole of these in soybeans
and soy foods.
Recent trends in sample preparation include automation, highthroughput performance, reduction in solvent volume and time,
on-line coupling with analytical instruments and more importantly, reduction of sample manipulation. Although signicant
advances on the extraction of isoavones using modern extraction techniques have been achieved, the full potential of the new
technology available needs to be further explored. There is increasing evidence that a combination of extraction/clean-up techniques
such UAE, PLE and SPE is the most promising application of the new
available technology for the development of extraction methods for
the determination of isoavones. Using such combinations not only
high-throughput performance and on-line coupling with the analysis instrument is possible, but also the reduction of post-extraction
steps necessary before analysis. Moreover, the availability of new
SPE sorbents may be easily used in the future to improve the performance of developed combined methods using actual available
technologies. Another advantage of the use of PLE is the possibility
of re-extraction of the same sample without manipulation which
reduces analytical errors. This characteristic is very interesting
since it may be recommendable to perform sequential extractions
(up to 5 extractions of the same sample) in order to ensure that
quantitative extractions are achieved. For the extraction procedure,
the natural tendency is to use less toxic, expensive and environmental friendly solvents, such as ethanol, under optimized conditions
that maximize extraction efciency achieving fast and quantitative
recoveries.
Another technical tendency in sample preparation is to minimize pre and post-extraction steps in order to take full advantage
of fast extraction and analysis procedures and to reduce analytical errors due to sample manipulation. This trend coupled to the
increasing availability of commercial standards and higher sensitivity and resolution of new chromatography technology points
toward the use of non-hydrolytic methods for the quantication of
soy isoavones. Furthermore, gathering full information about the
distribution and concentration of all main soy isoavone chemical
forms present in the samples may be critical to understand the role
of these compounds in preventing diseases.
Similarly, for the determination of isoavones the current trend
is toward fast, high sensitive and high-resolution separation of all
main chemical forms of these compounds in soybeans and soy
foods. The development of new column packing technology and
materials as well as of chromatographic systems that can operate at high pressures allows analysis time to be drastically reduced
from the usual 60 min to a few minutes with outstanding performances showing that further advances can be made in analytical
methodology currently used.
Altogether, the optimization of sampling, sample preservation
and sample preparation parameters are critical for accurate estimation of isoavones present in soybeans and soy foods. Accurate
estimation of isoavones in soybeans and soy foods, as well as in
other samples, will enable researchers to correctly evaluate the
inuence of such phytochemicals on health and provide precise
28
dietary and safety guidelines on consumption of these compounds.

In addition, accurate quantication of isoavones in soybeans and
soy foods will allow manufacturers, consumers, and marketing professionals to differentiate quality value-added products from the
conventional ones. Most importantly, the scientic community,
including journal editors and referees, must recognize the necessity
of validating sampling, preservation and extraction as critical steps
in describing the content of isoavones in foods, in order to advance
in the eld of functional foods for both research and industry.
Acknowledgment
The authors acknowledge funding from the Instituto Nacional
de Investigacin y Tecnologa Agraria y Alimentaria (INIA) project
AT07-003.
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Journal of Chromatography A, 1216 (2009) 229

Contents lists available at ScienceDirect

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Fig. 1. Chemical structures of soybean isoavones and abbreviations.

ity (similar effects to estradiol hormones). The basic characteristic

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

When the intake of isoavones is estimated, the quality of the

In analytical practices, the importance of sample conservation

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Fig. 3. Most common possible degradation paths of soybean isoavones.

for all different conjugated forms to generate the aglycone forms

and inuence of soybean variety as observed by Kim et al. [75] may,

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Also, while studying isoavone proles and distribution in

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

as such. However, it is of crucial importance when using hydrolytic

extraction solvent, one of the key parameters of any extraction

Di, Gi, Gly, De, Ge and

Fixed extraction conditions

80% CH3 CN and 80% CH3 CN

The amount of water

Gi, Ge, Di and De

Solvent: 25 mL of pure solvent

Soy isolate, tofu, soybeans and miso

Ge, De, Gi, Di, Gly, MGi, MDi,

Toasted soy our

Di, Gi, Gly, De, Ge, Gle,

Di, Gi, Gly, De, Ge, Gle, MDi,

Technique: tumbling mixer

Technique: rotary mixer

Soy our, tempeh, TVP and soy germ

Di, Gi, Gly, De, Ge, Gle, MDi,

53% CH3 CN without

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Di, Gi, Gly, De, Ge, Gle, MDi,

58% CH3 CN, 58% CH3 CN (+0.1N

58% CH3 CN without

80% CH3 CNHCl 0.1N, 5

Defatted soybean meal, soy protein isolate

Di, Gi, Gly and MGi

Di, Gi, Gly, De, Ge, Gle, MDia ,

Di, Gi, Gly, MDi, MGly, MGi, De

Proposed method and

Solvent: 4099.99% EtOH

Extraction time: 224 h

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Another approach of solvent selection was given by Achouri et

(4099.99%), solvent volume to sample ratio (1:1 to 10:1 mL g1 ),

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

stirring for 2 h at room temperature. Another important remark

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

waves (UAE) and the physicalchemical changes in the solvent that

and without modications, for routine analysis, to obtain isoavone

M.A. Rostagno et al. / J. Chromatogr. A 1216 (2009) 229

Defatted soybean meal and

Di, Gi, Gly and MGi

Di, Gi, Gly, De, Ge,

50% EtOH, 60 C, 0.1 g, 20 min

80% MeOH and 80% EtOH,

80% CH3 CN (0.1N HCl)

Temperature: 2580 C (ultrasonic bath)